Down regulation of plant cyclin-dependent kinase inhibitors

ABSTRACT

The present invention provides isolated nucleic acid molecules encoding plant cyclin dependent kinase inhibitors (ICKs). The nucleic acid molecules may be used to downregulate expression of a plant ICK, thereby providing useful plant phenotypes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a section 371 application of PCT/IB01/01492, filedJun. 29, 2001, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Nos. 60/218,471, filed Jul. 14, 2000 and60/241,219, filed Oct. 13, 2000.

RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/218,471, filed Jul. 14, 2000 and to U.S. provisional patentapplication Ser. No. 60/241,219, filed Oct. 13, 2000. The contents ofthese provisional patent applications are incorporated herein byreference in their entirety.

BACKGROUND TO THE INVENTION

When eukaryotic cells, e.g., plant cells, divide they go through ahighly ordered sequence of events collectively termed as the “cellcycle.” Briefly, DNA replication or synthesis (S) and mitoticsegregation of the chromosomes (M) occur with intervening gap phases (G1and G2) and the phases follow the sequence G1-S-G2-M. Cell division iscompleted after cytokinesis, the last step of the M-phase. Cells thathave exited the cell cycle and have become quiescent are said to be inthe G0 phase. Cells at the G0 stage can be stimulated to re-enter thecell cycle at the G1 phase.

The transition between the different phases of the cell cycle isbasically driven by the sequential activation/inactivation of a kinase,known as “cyclin-dependent kinase” or “CDK” by different molecules.Required for kinase activation are proteins called cyclins which arealso important for targeting the kinase activity to a given (subset of)substrate(s). Other factors regulating CDK activity include CDKinhibitors (known as CKIs, ICKs, Kips, Cips, Inks, or KRPs, i.e.,Kip-related proteins), CDK activating kinase (CAK), CDK phosphatase(Cdc25), and CDK subunit (CKS) (Mironov et al. (1999) Plant Cell 11,509-522 and Reed (1996). Prog. Cell Cycle Res. 2, 15-27).

The existence of an inhibitor of mitotic CDKs was inferred fromexperiments with endosperm of maize seed (Grafi and Larkins (1995)Science 269, 1262-1264) but only recently were ICKs identified inplants. A total of seven Arabidopsis, one Chenopodium rubrum, and onealfalfa ICK cDNAs have to date been described. The encoded proteins arecharacterized by a stretch of approximately 35 carboxy-terminal aminoacids showing homology to the amino-terminal cyclin/Cdk binding domainof animal ICKs of the p21^(CiP1)/p27^(Kip1)/p57^(KiP2)-types. Outsidethe carboxy-terminal region, the plant ICKs are unrelated to one anotherand no homologies have been detected with other protein sequences.

Arabidopsis ICK1 is able to inhibit the kinase activity of plant Cdc2,but not the kinase activity of human Cdc2 or of S. cerevisiae Cdc28.Arabidopsis ICK3 also inhibits plant Cdc2 kinase activity. ICK1 and ICK3interact both with Arabidopsis Cdc2a (A-type Cdk) and cyclin D3, but notwith Arabidopsis Cdc2b (B-type Cdk). ICK1 also interacts with cyclin D1and cyclin D2, but not with cyclin A2, cyclin B1, cyclin B2, or PCNA. Asdetermined by yeast two-hybrid assays, interaction between ICK1 andcyclin D3 is much stronger than between ICK1 and Cdc2a Thecarboxy-terminal region of ICK1 (homologous to the Cip1/Kip1,2 animalICKs) is required for association with Cdc2a and cyclin D3. Binding toeither of these partners is, however, strongly enhanced with anamino-terminally deletion mutant comprising the approximately 50 aminoacid stretch upstream of the ICK1 cyclin/Cdk binding domain inconjunction with this domain. Wild-type ICK1 does not fit as tightly toCdc2a or cyclin D3 as the foregoing deletion mutant, suggesting thepresence of destabilizing elements in the amino-terminal region of ICK1.

ICK1 expression is highest in leaves and both abscisic acid andincubation at low temperature conditions, which inhibit plant celldivision, induce the accumulation of ICK1 transcripts in Arabidopsisseedlings. Expression of ICK2 is most prominent in stems and inflorescence apices, lowest in 1-week-old seedlings and upregulated bytreatment with 0.1% NaCl (WO9914331, Lui et al. (2000) Plant J. 21,379-385, Wang et al. (1997) Nature 386, 451-452, WO9964599).

Transgenic Arabidopsis, Brassica napus and B. carinata plants have beengenerated expressing ICK1 under the control of the AP2 promotersustaining pollen-preferred expression or under the control of theanther-specific Bgl1 promoter. Increased ICK1 mRNA levels in AP2-ICK1transgenic Arabidopsis plants (T1 and T2) correlated with phenotypiceffects ranging from no visible petals to visible petals of reduced sizeto normal petals. Only plants with normal petals are self-fertile. Seedsetting in the male sterile transgenic plants with the other phenotypescan be restored by fertilization with wild-type pollen. No significantmale sterility was observed in Bgl1-ICK1 transgenic Arabidopsis plants.The effects of AP2-ICK1 and Bgl1-ICK1 were less severe and morepronounced, respectively, in transgenic Brassica plants (WO9964599).

Many different functions have been described for ICKs of animal originand these include: differential inhibition of cyclin-Cdk kinaseactivity, regulation of cyclin-Cdk complex assembly, regulation ofcommitment of cells to divide by integrating mitogenic and antimitogenicsignals, regulation of cell cycle progression, regulation of DNAreplication and DNA repair, regulation of gene transcription, regulationof cyclin degradation, involvement in cell cycle withdrawal and celldifferentiation, regulation of apoptosis, control of organ and organismsize and regulation of endoreduplication (Nakayama and Nakayama 1998Bioesseys 20, 1020-1029). Many of these functions have been attributedto the ICK domains outside the cyclin/Cdk binding regions.

In view of the unusually pronounced sequence heterogeneity between theICKs of a single plant species and the differences in expressionpatterns, it can be expected that each of the plant ICKs serves a uniquefunction in controlling plant development. Such plant ICK functions mayinclude interference with cell cycle events similar as those regulatedby ICKs in animals but as yet unidentified in plants.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “Inhibitors of Cyclin DependentKinases” or “ICK” nucleic acid and polypeptide molecules. The ICKnucleic acid and polypeptide molecules of the present invention areuseful as modulating agents in regulating cell cycle progression in, forexample, plants. The ICK nucleic acid and polypeptide molecules of thepresent invention are particularly useful in agriculture and plant celland tissue culture.

In one aspect, the present invention features a method for modulatingthe cell cycle in a plant, e.g., arabidopsis thaliana, rice, wheat,maize, tomato, alfalfa, oilseed rape, soybean, sunflower, or canola, byintroducing into the plant an ICK modulator, such that the cell cycle inthe plant is modulated. In one embodiment, the plant is a monocot plant.In another embodiment, the plant is a dicot plant.

In one embodiment, the ICK modulator is a small molecule. In anotherembodiment, the ICK modulator is capable of modulating ICK polypeptideactivity. For example, the ICK modulator can be an anti-ICK antibody oran ICK polypeptide comprising the amino acid sequence of SEQ ID NO:10,11, 12, 13, 14, 15, 16, 17, 44, or 55, or a fragment thereof.

In another embodiment, the ICK modulator is capable of modulating ICKnucleic acid expression. For example, the ICK modulator can be anantisense ICK nucleic acid molecule, an ICK gene silencing molecule, aribozyme, or a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56, or a fragmentthereof.

In another aspect, the invention features a method for modulating plantgrowth and/or plant cell fate and/or plant morphology and/or plantbiochemistry and/or plant physiology by introducing into a plant an ICKmodulator, such that said plant growth and/or plant cell fate and/ormorphology and/or plant biochemistry and/or plant physiology ismodulated.

In a further aspect, the invention features a method for improvingtolerance to an environmental stress condition, e.g., drought, salt,temperature, or nutrient deprivation, in a plant by introducing into theplant an ICK modulator, such that tolerance to an environmental stresscondition in the plant is improved.

In another aspect, the invention features a method for improvingtolerance to a plant pathogen, e.g., a pathogenic bacterium such asAgrobacterium tumefaciens, plant pathogenic fungi includingPlasmodiophora brassicae, Crinipellis perniciosa, Pucciniastrumgeoppertianum, Taphrina wiesneri, Ustilaga maydis, Exobasidium vaccinii,E. camelliae, Entorrhiza casparyana and Apiosporina morbosum, thatabuses the cell cycle in a plant by introducing into the plant an ICKmodulator, such that tolerance to a plant pathogen that abuses the cellcycle in the plant is improved.

In a further aspect, the invention features a method for modulating ICKactivity in a plant by introducing into the plant an ICK modulator, suchthat ICK activity in the plant is modulated.

The present invention also features a method for modulating the cellcycle in a plant cell by contacting the plant cell with an ICKmodulator, such that the cell cycle in the plant cell is modulated.

In another aspect, the invention provides a method for modulating plantcell growth and/or plant cell fate and/or plant morphology and/or plantbiochemistry and/or plant physiology by contacting a plant cell with anICK modulator, such that plant cell growth and/or plant cell fate and/orplant morphology and/or plant biochemistry and/or plant physiology ismodulated.

Methods for improving tolerance to an environmental stress condition ina plant cell and methods for improving tolerance to a plant pathogenthat abuses the cell cycle in a plant cell are also provided by thepresent invention. These methods include contacting the plant cell withan ICK modulator, such that tolerance to an environmental stresscondition or to a plant pathogen that abuses the cell cycle in the plantcell is improved.

Methods for modulating ICK activity in a plant cell are also provided.The methods include contacting the plant cell with an ICK modulator,such that ICK activity in the plant cell is modulated.

In another embodiment the invention provides transgenic plants (e.g.,monocot or dicot plants) containing an isolated nucleic acid molecule ofthe present invention. For example, the invention provides transgenicplants containing a recombinant expression cassette including a plantpromoter operably linked to an isolated nucleic acid molecule of thepresent invention (e.g., a nucleic acid molecule comprising thenucleotide sequence set forth in SEQ ID NO:9, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:54 or SEQ ID NO:56). The present invention alsoprovides seed, pollen, cuttings, and flowers from the transgenic plants.In another embodiment the invention provides methods of modulating, in atransgenic plant, the expression of the nucleic acids of the invention.

Isolated nucleic acid molecules which include the nucleotide sequenceset forth in SEQ ID NO: 9, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:54 orSEQ ID NO:56, or complements thereof, are also encompassed by thepresent invention.

In one aspect, the invention features an isolated nucleic acid moleculewhich encodes an ICK polypeptide comprising the amino acid sequence setforth in SEQ ID NO:10, 11, 12, 13, 14, 15, 16, 17, 44, or 55, or acomplement of the isolated nucleic acid molecule.

In another aspect, the present invention features an isolated ICKpolypeptide comprising the amino acid sequence of SEQ ID NO:10, 11, 12,13, 14, 15, 16, 17, 44, or 55, or fragments thereof.

The proteins of the present invention or portions thereof, e.g.,biologically active portions thereof, can be operably linked to anon-ICK polypeptide (e.g., heterologous amino acid sequences) to formfusion proteins. The invention further features antibodies, such asmonoclonal or polyclonal antibodies, that specifically bind to the ICKpolypeptides of the invention. In addition, the ICK polypeptides orbiologically active portions thereof can be incorporated intopharmaceutical compositions, which optionally include pharmaceuticallyacceptable carriers.

In another aspect, the present invention provides a method foridentifying a compound which binds to an ICK polypeptide of the presentinvention. The method includes contacting the polypeptide, or a cellexpressing the polypeptide with a test compound; and determining whetherthe polypeptide binds to the test compound. In one embodiment, thebinding of the test compound to the polypeptide is detected by directdetection of test compound/polypeptide binding. In another embodiment,the binding of the test compound to the polypeptide is detected bydetection of binding using a competition binding assay. In yet anotherembodiment, the binding of the test compound to the polypeptide isdetected using an assay for ICK activity.

In yet another aspect, the present invention provides a method formodulating the activity of an ICK polypeptides of the present invention.The method includes contacting the polypeptide or a cell expressing thepolypeptide with a compound which binds to the polypeptide in asufficient concentration to modulate the activity of the polypeptide.

In a further aspect, the present invention provides a method foridentifying a compound which modulates the activity of an ICKpolypeptide of the present invention. The method includes contacting anICK polypeptide with a test compound; and determining the effect of thetest compound on the activity of the polypeptide to thereby identify acompound which modulates the activity of the polypeptide.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence of the full length OsICK2 cDNA(SEQ ID NO:9). The open reading frame (between rounded brackets) thestart codon and the stop codon (underlined) are indicated. Alsoindicated are the polyadenylation sites (double underlined), the AU-richelements or AREs (italic), and the extra 3′UTR region of SEQ ID NO:9compared to SEQ ID NO:1 (underlined between square brackets).

FIG. 2 depicts the nucleotide sequence of the full length OsICK4 cDNA(SEQ ID NO:43). The open reading frame (between rounded brackets) thestart codon and the stop codon (underlined) are indicated. Alsoindicated is the AU-rich element or ARE (italic).

FIG. 3 depicts an amino acid alignment of the full-length OsICK2 aminoacid sequence (SEQ ID NO:10), the full-length OsICK4 amino acid sequence(SEQ ID NO:43) and the predicted full-length OsICK5 amino acid sequence(SEQ ID NO:55) with the amino acid sequences of other known plant ICKs:the seven Arabidopsis ICKs (ICK1 to ICK7), the Medicago sativa ICK(alfalfa ICK), and the Chenopodium rubrum ICK (Chenopod ICK). Thealignment was made using ClustalW as part of the VNTI (version 5.5;InforMax Inc.) software using default parameter settings (blosum 62scoring matrix; gap opening penalties for pairwise and multiplealignment=10; gap extension penalty for pairwise alignment=0.1; gapextension penalty for multiple alignment=0.05). Identical amino acidresidues are indicated in black shaded boxes. Conserved amino acidresidues are indicated in grey shaded boxes according to the groups(M,I,L,V), (A,G,S,T), (R,K,H), (F,W,Y), (D,E) and (N, Q).

FIG. 4 depicts an amino acid alignment of the relevant parts of thefull-length OsICK2 amino acid sequence (SEQ ID NO:10), the full-lengthOsICK4 amino acid sequence (SEQ ID NO:43) and the predicted full-lengthOsICK5 amino acid sequence (SEQ ID NO:55) with the partial amino acidsequence of OsICK1 (SEQ ID NO:11), the partial amino acid sequence ofOsICK3 (SEQ ID NO:12), the partial amino acid sequence of the Zea maysICK1 (SEQ ID NO:14), the partial amino acid sequence of the Zea maysICK2 (SEQ ID NO:15), the partial amino acid sequence of the Sorghumbicolor ICK (SEQ ID NO:16), and the partial amino acid sequence of thePinus taeda ICK (SEQ ID NO: 17). The alignment was made using ClustalWas part of the VNTI (version 5.5; InforMax Inc.) software using defaultparameter settings (blosum 62 scoring matrix; gap opening penalties forpairwise and multiple alignment=10; gap extension penalty for pairwisealignment=0.1; gap extension penalty for multiple alignment=0.05).Identical amino acid residues are indicated in black shaded boxes.Conserved amino acid residues are indicated in grey shaded boxesaccording to the groups (M,I,L,V), (A,G,S,T), (R,K,H), (F,W,Y), (D,E)and (N, Q).

FIG. 5 is a schematic representation of the motifs conserved in plantICK molecules (using the same numbering indicated in Table 1). Furtherindicated in FIG. 4 are the secondary structure elements predicted to bepresent in the plant ICK molecules. (H: predicted α-helical structure ina stretch of at least 4 consecutive amino acid residues. E: predictedextended β-sheet structure in a stretch of at least 4 consecutive aminoacids).

FIG. 6 depicts the Cy-box-regions present in different mammalianproteins and the derived consensus sequence. p21Cip1, p27Kip1 andp57kip2 are three types of mammalian cyclin-dependent kinase inhibitors.Cdc25A is a G1/S specific protein phosphatase which has Cdc2-relatedproteins as substrates. p107 belongs to the family of pocket proteinsalso including p130 and pRB. E2F1 is a transcriptional activatorinvolved in activation of S-phase specific gene expression. X may be anyamino acid and H may be M,I,L or V.

FIG. 7 is a blot depicting the expression of OsICK1 (‘ICK1’), OsICK2(‘ICK2’) and OsICK4 (‘ICK4’) in different tissues of rice plants. cDNAobtained by RT-PCR was subjected to DNA gel blot analysis withbiotin-labeled probes specific for OsICK1, OsICK2 and OsICK4. (R: roottissue, L: leaf tissue, STM: stem meristem tissue, ST: stem tissue, S:seeds).

FIG. 8 is a blot depicting the expression of OsICK1 (‘ICK1’), OsICK2(‘ICK2’) and OsICK4 (‘ICK4’) in developing rice grains. cDNA obtained byRT-PCR was subjected to DNA gel blot analysis with biotin-labeled probesspecific for OsICK1, OsICK2 and OsICK4. (DAP: days after pollination).

FIG. 9 depicts the results from an in situ hybridization analysis ofOsICK2 expression in rice seeds collected 7 days after pollination. Darkfield microscopy (right panel) visualizes a clear (bright white)hybridization signal in the cell layer(s) surrounding the developingendosperm (white arrow). A patchy hybridization signal is also obviousin the developing embryo (white arrow). The bright field microscopicimage (left panel) shows the morphology of the toluidine blue-stainedsection.

FIGS. 10A-I depicts the results from an in situ hybridization analysisof OsICK2 expression in rice seeds collected 20 days after pollination.(A) and (B) are dark field and bright field microscopic images,respectively, of the same longitudinal section. (C) is a magnificationof a part of (A). Clearly visible is the (bright white) hybridizationsignal in the scutellum (white arrow). (D) and (E) are dark field andbright field microscopic images, respectively, of the same crosssection. (F) is a magnification of a part of (D). Hybridization signalsin (D) and (F) show expression of OsICK2 in the cell layer(s) lining thedeveloping endosperm (white arrows). (G) and (H) are dark field andbright field microscopic images, respectively, of the same longitudinalsection again showing OsICK2 expression in the cell layers surroundingthe developing endosperm (white arrow). The same is shown in the brightfield microscopic image (I) where the hybridization signal is black(black arrow).

FIG. 11 depicts A comparison of the OsICK4 cDNA sequences (Panel A) andOsICK4 protein sequences (Panel B) obtained by (i) yeast two-hybridinteraction screening of rice two-hybrid cDNA library using Cdc2-Os1 asbait and by (ii) hybridization screening of the same library. Thefull-length OsICK4 cDNA sequence obtained by hybridization screening isgiven in Panel A with the ORF between brackets. Further indicated inPanel A is the part of the OsICK4 cDNA missing in the clone identifiedby yeast two-hybrid interaction cloning, said part being indicated inunderlined italics. The corresponding full-length OsICK4 amino acidsequence is given in Panel B where it is identified as “HYB ICK4”.Further indicated in Panel B is the part of the amino acid sequenceencoded by the partial OsICK4 clone as outlined in Panel A. The partialamino acid sequence is identified in Panel B as “2-H ICK4”. Amino acidscommon to both proteins are shaded in grey.

FIG. 12 shows the wrongly predicted OsICK4 protein sequence present inGenBank (GenBank accession number AC069145 version 5 of Sep. 30, 2000;protein ID=AAG16867.1). The 48 superfluous amino acids in the predictedprotein sequence not present in the protein sequence derived (SEQ IDNO:44) from the experimentally obtained OsICK4 cDNA (SEQ ID NO:43) areindicated between square brackets and as underlined boldface characters.

FIG. 13 depicts the binary plant transformation vector p0428 ICK4comprising the OsICK4 ORF operably linked to the GOS2 promoter.

FIG. 14 depicts the pUC18 derivative vector pUC18-ICK4 CS comprising thecassette with inverted repeats of an OsICK4 cDNA fragment separated bythe tobacco MAR sequence. This cassette is used for co-suppression ofOsICK4 expression in transgenic plants.

FIG. 15 depicts the binary plant transformation vector p0490 comprisingthe OsICK4 co-suppression cassette of pUC-ICK4 CS (see FIG. 13) operablylinked to the GOS2 promoter.

FIG. 16 depicts the binary plant transformation vector p0489 comprisingthe OsICK4 co-suppression cassette of pUC-ICK4 CS (see FIG. 13) operablylinked to the prolamine promoter.

FIG. 17 depicts the binary plant transformation vector p0488 comprisingthe OsICK4 co-suppression cassette of pUC-ICK4 CS (see FIG. 13) operablylinked to the oleosine promoter.

FIG. 18 depicts the binary plant transformation vector p0559 comprisingthe OsICK4 co-suppression cassette of pUC-ICK4 CS (see FIG. 13) operablylinked to the gluteline promoter.

FIG. 19 depicts the genomic fragment corresponding to the full-lengthOsICK4 cDNA.

FIG. 20 depicts the genomic fragment corresponding to the predictedfull-length OsICK5 cDNA. The depicted fragment (SEQ ID NO:56)corresponds to the inverse complement of nucleotides 6331 to 7403 ofGenBank accession number AP003525.1 as available on Jun. 26, 2001.Indicated in grey shaded boxes are the positions of the primers (SEQ IDNO:57 and 58) used to amplify the OsICK5 genomic fragment.

FIG. 21 depicts the predicted full-length OsICK5 cDNA sequence (SEQ IDNO:54; panel A) and the full-length OsICK5 protein sequence deducedthereof (SEQ ID NO:55; panel B). Uncertain residues in the predictedcDNA are indicated by ‘N’ and marked by a grey shaded box. Thetranslation of the uncertain nucleotide residues in the cDNA result inan uncertain amino acid residue ‘X’ in the deduced protein sequence alsomarked by a grey shaded box. Further indicated in panel (A) in greyshaded boxes are the positions of the primers (SEQ ID NO:57 and 58) usedto amplify the OsICK5 cDNA. Indicated in black shaded boxes are thestart and the stop codon.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “Inhibitors of Cyclin DependentKinases” or “ICK” nucleic acid and polypeptide molecules. In particular,the present invention is based on the discovery of the firstmonocotyledonous (monocot), e.g., cereal, ICK molecule. The ICKmolecules of the present invention were identified by database miningusing the “GRYEW” amino acid motif as a query sequence. This motif ispresent at the carboxyl-terminus of all known plant ICK molecules.

As used herein, the terms “Inhibitors of Cyclin Dependent Kinases” and“ICK” include molecules of the present invention which are capable ofinhibiting the activity of a Cyclin Dependent Kinase (CDK), e.g., aplant CDK. CDKs are a group of serine/threonine kinases which regulatethe progression of the cell cycle in eukaryotes, e.g., plants. CDKs aretypically complexed with cyclins forming an enzyme complex, CDK beingthe catalytic subunit and cyclin being the regulatory subunit of theenzyme complex (Wang, H. (1997) The Plant Journal 15(4):501-510). ICKmolecules of the present invention, e.g., ICK molecules from a monocotplant such as rice, typically have one or more of the followingactivities: inhibition of CDK activity (e.g., cyclin-CDK activity);regulation of cyclin-CDK complex assembly; regulation of the commitmentof cells to divide, e.g., by integrating mitogenic and antimitogenicsignals; regulation of cell cycle progression; regulation of DNAreplication and/or DNA repair; they regulate gene transcription;regulation of cyclin degradation; involvement in cell cycle withdrawaland/or cell differentiation; regulation of cell death, e.g., apoptosis;control of organ (e.g., plant organ) and/or organism (e.g., plantorganism) size; and regulation of endoreduplication.

As used herein, the term “cell cycle” includes the cyclic biochemicaland structural events associated with growth, division, andproliferation of cells, and in particular with the regulation of thereplication of DNA and mitosis. The cell cycle is divided into periodscalled: G₀, Gap₁ (G₁), DNA synthesis (S), Gap₂ (G₂), and mitosis (M).Normally these four phases occur sequentially, however, the term cellcycle also includes modified cycles wherein one or more phases areabsent resulting in a modified cell cycle such as endomitosis,acytokinesis, polyploidy, polyteny, and endoreduplication.

As used herein, the term “plant” includes whole plants, plant organs(e.g., leaves, stems, or roots), plant tissue, plant seeds, and plantcells and progeny thereof. The class of plants which can be used in themethods of the invention is generally as broad as the class of higherplants amenable to transformation techniques, including bothmonocotyledonous and dicotyledonous plants. Particularly preferredplants are Arabidopsis thaliana, rice, wheat, barley, sorghum, maize,tomato, potato, cotton, alfalfa, oilseed rape, soybean, cotton,sunflower or canola. The term plant also includes monocotyledonous(monocot) plants and dicotyledonous (dicot) plants including a fodder orforage legume, ornamental plants, food crops, trees, or shrubs selectedfrom the list comprising Acacia spp., Acer spp., Actinidia spp.,Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor,Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans,Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp.,Bruguiera gymnorrhiza, Burkea africana, Butea fronuosa, Cadaba farinosa,Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassiaspp., Centroema pubescens, Chaenomneles spp., Cinnamomum cassia, Coffeaarabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina,Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydoniaoblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata,Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodiumspp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp,Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp.,Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp.,Euclea schimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana,Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium thunbergii,Ginkgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum,Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemarthiaaltissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa,Hypericum erectum, Hyperthelia dissoluta, Indigo incarnata, Iris spp.,Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaenaleucocephala, Loudetia simplex, Lotonus bainesii, Lotus spp.,Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa,Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp.,Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum,Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp.,Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca,Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria fleckii,Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudotsugamenziesii, Pterolobium stellatum, Pyrus communis, Quercus spp.,Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribesgrossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp.,Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoiasempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp.,Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthios humilis,Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp.,Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitisvinifeia, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays,amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage,canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil,oil seed rape, okra, onion, potato, rice, soybean, straw, sugarbeet,sugar cane, sunflower, tomato, squash, and tea, amongst others, or theseeds of any plant specifically named above or a tissue, cell or organculture of any of the above species.

The term “plant cell”, as used herein includes seeds, e.g., seedsuspension cultures, embryos, cells from meristematic regions, cellsfrom callus tissue, cells from leaves, cells from roots, cells fromshoots, gametophytes, sporophytes, pollen, and microspores.

The ICK molecules of the present invention are involved in cell cycleregulation in plants. Accordingly, the ICK molecules of the presentinvention, or derivatives thereof, may be used to modulate the cellcycle in a plant by, for example, modulating the activity or level ofexpression of ICK; altering the rate of the cell cycle or phases of thecell cycle; or altering entry into and out of the various cell cyclephases. In plants, the ICK molecules of the present invention may beused in agriculture to, for example, improve the growth characteristicsof a plant such as the growth rate of a plant; the size of specifictissues or organs in a plant; or the architecture or morphology of aplant. The ICK molecules of the present invention may also be used inagriculture to increase crop yield, improve tolerance to environmentalstress conditions (such as drought, salt, temperature, or nutrientdeprivation), improve tolerance to plant pathogens that abuse the cellcycle, or as targets to facilitate the identification of inhibitors oractivators of ICKs that may be useful as herbicides or plant growthregulators.

As used herein, the term “cell cycle associated disorders” includes adisorder, disease or condition which is caused or characterized by amisregulation (e.g., downregulation or upregulation), abuse, arrest, ormodification of the cell cycle. In plants cell cycle associateddisorders include endomitosis, acytokinesis, polyploidy, polyteny, andendoreduplication which may be caused by external factors such aspathogens (nematodes, viruses, fungi, or insects), chemicals,environmental stress (e.g., drought, temperature, nutrients, or UVlight) resulting in, for example, neoplastic tissue (e.g., galls, rootknots) or inhibition of cell division/proliferation (e.g., stuntedgrowth).

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as ICK protein and nucleic acidmolecules, which comprise a family of molecules having certain conservedstructural and functional features. The term “family” when referring tothe protein and nucleic acid molecules of the invention is intended tomean two or more proteins or nucleic acid molecules having a commonstructural domain or motif and having sufficient amino acid ornucleotide sequence homology as defined herein. Such family members canbe naturally or non-naturally occurring and can be from either the sameor different species. For example, a family can contain a first proteinof plant, e.g., rice, origin, as well as other, distinct proteins ofplant, e.g., rice, origin or alternatively, can contain homologues ofother plant, e.g. wheat or maize, or of non-plant origin. Members of afamily may also have common functional characteristics.

In one embodiment of the invention, an ICK protein of the presentinvention, e.g., an ICK protein from a monocot plant such as rice, isidentified based on the presence of at least one or more of thefollowing motifs:

-   -   Motif 1: FXXKYNFD (SEQ ID NO:18), wherein X is any amino acid    -   Motif 2: [PL/]LXGRYEW (SEQ ID NO:19), wherein X is any amino        acid and [P/L] means that either a proline or a leucine appear        at the indicated position    -   Motif 3: EXE[D/E]FFXXXE (SEQ ID NO:20), wherein X is any amino        acid and [D/E] means that either an aspartate or a gluatamate        appear at the indicated position    -   Motif 4: YXQLRSRR (SEQ ID NO:21), wherein X is any amino acid    -   Motif 5: MGKY[M/I][K/R]KX[K/R] (SEQ ID NO:22), wherein X is any        amino acid, [M/I] means that either a methionine or an        isoleucine appear at the indicated position, and [K/R] means        that either a lysine or an arginine appear at the indicated        position    -   Motif 6: SXGVRTRA (SEQ ID NO:23), wherein X is any amino acid        (The foregoing motifs are summarized in Table 1 and graphically        represented in FIG. 4).

In a preferred embodiment, an ICK molecule of the present invention,e.g., an ICK molecule from a monocot plant such as rice, contains two,three, four, five, or, more preferably, all six of the foregoing motifs.All six motifs are present in the full-length OsICK2 (SEQ ID NO:10),full-length OsICK4 (SEQ ID NO:44) and predicted full-length OsICK5 (SEQID NO:55) proteins of the present invention. Although motifs 2 and 5 arepresent in the predicted full-length OsICK5 protein, they deviate fromthe consensus sequences.

Motifs 1, 2, and 3 are typically found in the carboxyl-terminal regionof plant ICK proteins. This region is believed to be involved in theinteraction of ICKs with both CDKs and cyclins (Chen et al. (1996) Mol.Cell Biol 16, 4673-4682, Matsuoka et al. (1995) Genes Dev. 9, 650-662,and Nakayama and Nakayama (1998) Bioessays 20, 1020-1029). Motifs 4, 5,and 6 are typically found in the amino-terminal region of plant ICKproteins.

TABLE 1 Conserved motifs in plant ICK proteins. ICK1 to ICK7 denote theArabidopsis thaliana ICKs. Motif 1 Motif 2 Motif 3 Motif 4 Motif 5 Motif6 Alfalfa 198- 211- 182- 74- 1- 45- ICK FMEKYNFD PLPGRYET EFEEFCAKHEYLQLRNRR MGRYMKKLK SDGVRTRA ICK1 167- 180- 151- 20- AC003040 FKKKYNFDPLEGRYEW EIEOFEVEAE YMQLRSRR ICK2 183- 197- 164- AL132979 CSMKYNFDLGGGRYEW ELEDFFQVAE ICK3 197- 210- 181- 58- 1- 26- AB012242 FMEKYNFDPLSGRYEW EMEEFFAYAE YLQLRSRR MGKYMKKSK SPGVRTRA ICK4 264- 277- 248- 102-1- 44- AC003974 FIEKYNFD PLPGRFEW EMDEFFSGAE YLQLRSRR MGKYIRKSK SLGVLTRAICK5 164- 177- 148- 54- 1- 24- AB028609 FIQKYNFD PLPGRYEW EIEDFFASAEYLQLRSRR MGKYIKKSK ALGFRTRA ICK6 173- 186- 155- AP000419 FIEKYNFDPLEGRYKW EIEDLFSELE ICK7 170- 183- 154- AG011807 FTEKYNYD PLEGRYQWELDDFFSAAE Chenopodium 171- 184- 155- 25- ICK FSEKYWFD PLKGRYDWEIEEFFAVAE IPQLRSRR AJ002173 OsICK2 233- 247- 217- 75- 1- 24- FAAKYNFDLDAGRFEW EIEAFFAAAE YLQLRSRM MGKYMRKFR VVGVRTRS OsICK1 ----YNYD PLQGRYEWOsICK3 FAEKY--- EIEAFFAAAE OsICK4 170- 183- 154- 48- 1- 28- FIDKYNFDPLPGRFEW ELEAFFAAEE YLELRSRR MGKYMRKAK PLGVRTRA OsICK5 196- 209- 180-63- 1- 20- FAAKYNFD PLDAGGAGRF EIEEFLAAAE YLRLRSRR MGKKKRDG VGGVRTRA EWZmICK1 FASKYNFD LDAGRFEW EIQEFFAAAE ZmICK2 FIDEYNFD PLPGRFEW EMNEYFAAEQSbICK  FAEAYNYD PLEGRFEW EIEAFAAAE CONSENSUS FX₂KYNFD (P/L)LXGREXE(D/E)FFX₃E YXGLRSRR MGKY[M/I] SXGVRTRA [Y/F]EW [K/R]KX[K/R]

The ICK proteins of the present invention from a monocot plant such asrice, in particular, are characterized by extensive α-helical stretchesespecially in between motifs 5 and 6 and in between motifs 6 and 4.Furthermore, in the ICK proteins of the present invention from a monocotplant such as rice, the region between motifs 4 and 3, only containspredicted α-helical segments and no extended β-sheets. These secondarystructure characteristics of the ICK proteins from a monocot plant suchas rice, are different from those found in alfalfa ICK and ArabidopsisICKs ICK3, ICK4 and ICK5.

In another embodiment of the invention, an ICK protein of the presentinvention, e.g., an ICK protein from a monocot plant such as rice, isidentified based on the presence of a “Cy-box.” As used herein, the term“Cy-Box” includes an amino acid sequence of about 5 amino acid residuesin length having the consensus sequence RXHuF (SEQ ID NO:24), wherein Xis any amino acid and Hu is a hydrophobic uncharged amino acid, such asM, I, L or V (see FIG. 5). Cy-boxes are typically involved in theinteraction of ICKs with cyclins. Amino acid residues 81-84 of theOsICK2 protein (SEQ ID NO: 10) are predicted to comprise a Cy-box(RMLF).

In another embodiment of the invention, an ICK protein of the presentinvention, e.g., an ICK protein from a monocot plant such as rice, isidentified based on the presence of a “nuclear localization sequence.”As used herein, the term “nuclear localization sequence” includes anamino acid sequence of about 4-20 amino acid residues in length, whichserves to direct a protein to the nucleus. Typically, the nuclearlocalization sequence is rich in basic amino acids, such as arginine (R)and lysine (K). A nuclear localization signal may have one or more ofthe sequences depicted in Table 2. Nuclear localization signals aredescribed in, for example, Gorlich D. (1998) EMBO 5.17:2721-7, thecontents of which are incorporated herein by reference. Amino acidresidues 54-57 of the OsICK2 protein (SEQ ID NO: 10) comprise a nuclearlocalization sequence. The Os ICK4 protein (SEQ ID NO:44) comprisesmultiple nuclear localization sequences as indicated in Table 2. Alsothe predicted full-length OsICK5 (SEQ ID NO:55) protein comprises anamino-terminal nuclear localization region (see Table 2).

TABLE 2 Potential nuclear localization sequences (NLSs) identified inplant ICKs using the PSORT/Prediction of protein localization sitessoftware (http://psort.nibb.ac.jp). NLS type Robbins & ICK 4-residuepattern Dingwall consensus Alfalfa ICK 80^(a)-RRLKRPLIRQHSAKRNKChenopodium 15-KKVSKSSYNIPQLRSRR ICK ICK2 23-KRRK ICK4 123-KRRK108-RRLQKKPPIVVIRSTKR 240-HRRR 112-KKPPIVVIRSTKRRKQQ 241-RRRP ICK560-RRLVKLPLLTNTRKQQK ICK7 5-KPKR 142-KKKK OsICK2^(b) 54-RRRK OsICK53-KKKK 4-KKKR ^(a)Position of the first amino acid of the site in theamino acid sequence of the indicated ICK. ^(b)Identification of theOsICK2 NLSs was done manually, i.e., the protein sequence was notanalyzed using the public PSORT software on the indicated Website.PSORT uses the following two rules to detect it: 4 residue patterncomposed of basic amino acids (K or R), or composed of three basic aminoacids (K or R) and H or P; a pattern starting with P and followed within3 residues by a basic segment containing 3 K or R residues out of 4residues. Another type of nuclear targeting signal is the type ofXenopus nucleoplasmin proposed by Robbins et al. (J. Robbins, S. M.Dilworth, R. A. Laskey, and C. Dingwall, Cell, 64, 615, 1991). Thepattern is: 2 basic residues, 10 residue spacer, and another basicregion consisting of at least 3 basic residues out of 5 residues.

In a further embodiment of the invention, an ICK protein of the presentinvention, e.g., an ICK protein from a monocot plant such as rice, isidentified based on the presence of a “PEST sequence.” As used herein,the term “PEST sequence” includes an amino acid sequence which isenriched in the amino acid residues proline (P), glutamate (E), serine(S) and threonine (T) and which is present in proteins with a highproteolytic turnover rate. PEST sequences are described in, for example,Rogers et al. (1986) Science 234, 364-368, the contents of which areincorporated herein by reference. Amino acid residues 167-191 of theOsICK2 protein, amino acid residues 117-131 of the OsICK4 protein andamino acid residues 128-145 of the predicted full-length OsICK5 proteincomprise potential PEST sequences (see Table 3).

TABLE 3 Potential PEST sequences identified in the plant ICKs using thePESTFIND software downloaded from http://ebi.ac.uk and run on a localserver. ICK Potential PEST sequences PEST score Alfalfa ICK11^(a)-KSESPSPNSTPTPSPSPSPTPITTNSPPPTTPNSSDGVR +24.12 Chenopodium105-RTADPEVESGEASSK +11.43 ICK ICK2 71-RDSPPVEEQCQIEEEDSSVSCCSTSEEK+15.46 ICK4 243-RPTTPEMDEFFSGAEEQQK +9.21 ICK5 100-KLEPDTTTEEACGDNER+13.68 ICK6 24-KLNDSSDSSPDSH +12.76 118-KETSPVSEGLGETTTEMESSSATK +15.73149-KTPTAAEIEDLFSELESQDDK +8.59 OsICK2 167-RETTPSSFLPGEVSDLESDLAGGQK+4.75 OsICK4 117-RDPDTISTPGSTTR +13.74 OsICK5 128-RPPGDADSSDAESNQEAK+13.12 ^(a)Position of the first amino acid of the site in the aminoacid sequence of the indicated ICK.

Isolated ICK proteins of the present invention, e.g., ICK proteins froma monocot plant such as rice, have an amino acid sequence sufficientlyidentical to the amino acid sequence of SEQ ID NO:10, 11, 12, 13, 14,15, 16, 17, 44 or 55, or are encoded by a nucleotide sequencesufficiently identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45,54 or 56. As used herein, the term “sufficiently identical” refers to afirst amino acid or nucleotide sequence which contains a sufficient orminimum number of identical or equivalent (e.g., an amino acid residuewhich has a similar side chain) amino acid residues or nucleotides to asecond amino acid or nucleotide sequence such that the first and secondamino acid or nucleotide sequences share common structural domains ormotifs and/or a common functional activity. For example, amino acid ornucleotide sequences which share common structural domains have at least30%, 40%, or 50% identity, preferably 60% identity, more preferably70%-80%, and even more preferably 90-95% identity across the amino acidsequences of the domains and contain at least one and preferably twostructural domains or motifs, are defined herein as sufficientlyidentical. Furthermore, amino acid or nucleotide sequences which shareat least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or90-95% identity and share a common functional activity are definedherein as sufficiently identical.

As used interchangeably herein, an “ICK activity”, “biological activityof ICK” or “functional activity of ICK”, refers to an activity exertedby an ICK protein, polypeptide or nucleic acid molecule on an ICKresponsive cell or tissue, or on an ICK protein substrate, as determinedin vivo, or in vitro, according to standard techniques. In oneembodiment, an ICK activity is a direct activity, such as an associationwith an ICK-target molecule, e.g., a CDK or a cyclin molecule. Suchinteractions have been experimentally demonstrated in the currentinvention. As used herein, a “target molecule” or “binding partner” is amolecule with which an ICK protein binds or interacts in nature, suchthat ICK-mediated function is achieved. An ICK target molecule can be anon-ICK molecule, e.g., a CDK or a cyclin molecule, or an ICK protein orpolypeptide of the present invention. In an exemplary embodiment, an ICKtarget molecule is an ICK ligand. Alternatively, an ICK activity is anindirect activity, such as a cellular signaling activity mediated byinteraction of the ICK protein with an ICK ligand. The biologicalactivities of ICK are described herein. For example, the ICK proteins ofthe present invention can have one or more of the following activities:(1) they may inhibit CDK activity (e.g., cyclin-CDK activity); (2) theymay regulate cyclin-CDK complex assembly; (3) they may regulate thecommitment of cells to divide, e.g., by integrating mitogenic andantimitogenic signals; (4) they may regulate cell cycle progression; (5)they may regulate DNA replication and/or DNA repair; (6) they mayregulate gene transcription; (7) they may regulate cyclin degradation;(8) they may be involved in cell cycle withdrawal and/or celldifferentiation; (9) they may regulate cell death, e.g., apoptosis; (10)they may control organ (e.g., plant organ) and/or organism (e.g., plantorganism) size; and (11) they may regulate endoreduplication.

Accordingly, another embodiment of the invention features isolated ICKproteins and polypeptides having an ICK activity. Preferred proteins areICK proteins, e.g., ICK proteins from a monocot plant such as rice,having at least one or more of the following domains: a motif 1, a motif2, a motif 3, a motif 4, a motif 5, a motif 6, a Cy-box, a nuclearlocalization sequence, or a PEST sequence, extensive α-helicalstretches, e.g. in between motifs 5 and 6, in between motifs 6 and 4,and in between motifs 4 and 3, and, preferably, an ICK activity.

Additional preferred proteins, e.g., ICK proteins from a monocot plantsuch as rice, have at least one or more of the following domains: amotif 1, a motif 2, a motif 3, a motif 4, a motif 5, a motif 6, aCy-box, a nuclear localization sequence, or a PEST sequence, extensiveα-helical stretches, e.g., in between motifs 5 and 6, in between motifs6 and 4, and in between motifs 4 and 3, and are, preferably, encoded bya nucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 43, 45, 54 or 56.

The sequences of the present invention are summarized below, in Table 4.

TABLE 4 PROTEIN CLONE ORGANISM DNA SEQUENCE SEQUENCE OsICK1 Oryza sativa2 11 (AQ574895) OsICK2 Oryza sativa 1 10 (partial length) 9 (fulllength) OsICK3 Oryza sativa 3 12 (AQ365042) OsICK4 Oryza sativa 4 13(AC069145) (partial length) (partial length) 43  44 (full length) (fulllength) 45  (genomic fragment) OsICK5 Oryza sativa 54  55 (AP003525)(predicted cDNA) (predicited 56  protein) (genomic fragment) ZmICK1 Zeamays 5 14 (AI737717) ZmICK2 Zea mays 6 15 (AW267370) SbICK Sorghumbicolor 7 16 (AF061282) PtICK Pinus taeda 8 17 (AA556411)

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in agricultural methods and screeningassays. The isolated nucleic acid molecules of the invention can beused, for example, to express an ICK protein (e.g., via a recombinantexpression vector in a host cell), to detect ICK mRNA (e.g., in abiological sample) or a genetic alteration in an ICK gene, and tomodulate ICK activity, as described further below. The ICK proteins canbe used to treat disorders characterized by insufficient or excessiveproduction of an ICK substrate or production of ICK inhibitors. Inaddition, the ICK proteins can be used to screen for naturally occurringICK substrates, to screen for drugs or compounds which modulate ICKactivity, as well as to treat disorders characterized by insufficient orexcessive production of ICK protein or production of ICK protein formswhich have decreased or aberrant activity compared to ICK wild typeprotein. Moreover, the anti-ICK antibodies of the invention can be usedto detect and isolate ICK proteins, regulate the bioavailability of ICKproteins, and modulate ICK activity.

A. Agricultural Uses:

In another embodiment of the invention, a method is provided formodifying cell fate and/or plant development and/or plant morphologyand/or biochemistry and/or physiology comprising the modification ofexpression in particular cells, tissues or organs of a plant, of agenetic sequence encoding an ICK, e.g., an ICK operably connected with aplant-operable promoter sequence.

Modulation of the expression in a plant of an ICK or a homologue,analogue or derivative thereof as defined in the present invention canproduce a range of desirable phenotypes in plants, such as, for example,the modification of one or more morphological, biochemical, orphysiological characteristics including: (i) modification of the lengthof the G1 and/or the S and/or the G2 and/or the M phase of the cellcycle of a plant; (ii) modification of the G1/S and/or S/G2 and/or G2/Mand/or M/G1 phase transition of a plant cell; (iii) modification of theinitiation, promotion, stimulation or enhancement of cell division; (iv)modification of the initiation, promotion, stimulation or enhancement ofDNA replication; (v) modification of the initiation, promotion,stimulation or enhancement of seed set and/or seed size and/or seeddevelopment; (vi) modification of the initiation, promotion, stimulationor enhancement of tuber formation; (vii) modification of the initiation,promotion, stimulation or enhancement of fruit formation; (viii)modification of the initiation, promotion, stimulation or enhancement ofleaf formation; (ix) modification of the initiation, promotion,stimulation or enhancement of shoot initiation and/or development; (x)modification of the initiation, promotion, stimulation or enhancement ofroot initiation and/or development; (xi) modification of the initiation,promotion, stimulation or enhancement of lateral root initiation and/ordevelopment; (xii) modification of the initiation, promotion,stimulation or enhancement of nodule formation and/or nodule function;(xiii) modification of the initiation, promotion, stimulation orenhancement of the bushiness of the plant; (xiv) modification of theinitiation, promotion, stimulation or enhancement of dwarfism in theplant; (xv) modification of the initiation, promotion, stimulation orenhancement of senescence; (xvi) modification of stem thickness and/orstrength characteristics and/or wind-resistance of the stem and/or stemlength; (xvii) modification of tolerance and/or resistance to bioticstresses such as pathogen infection; and (xviii) modification oftolerance and/or resistance to abiotic stresses such as drought stressor salt stress.

Methods to effect expression of an ICK or a homologue, analogue orderivative thereof as defined in the present invention in a plant cell,tissue or organ, include either the introduction of the protein directlyto a cell, tissue or organ such as by microinjection of ballistic meansor, alternatively, introduction of an isolated nucleic acid moleculeencoding the protein into the cell, tissue or organ in an expressibleformat. Methods to effect expression of an ICK or a homologue, analogueor derivative thereof as defined in the current invention in wholeplants include regeneration of whole plants from the transformed cellsin which an isolated nucleic acid molecule encoding the protein wasintroduced in an expressible format.

The present invention clearly extends to any plant produced by theinventive method described herein, and any and all plant parts andpropagules thereof. The present invention extends further to encompassthe progeny derived from a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by the inventivemethod, the only requirement being that the progeny exhibits the samegenotypic and/or phenotypic characteristic(s) as those characteristic(s)that (have) been produced in the parent by the performance of theinventive method.

Exploiting plant ICK functions to regulate plant growth and developmentcan depend on methods comprising enhancing ICK gene expression orectopic expression of ICK genes. For example, a method for exploitingplant ICK function comprises suppression of ICK gene expression, thus,relieving negative control on cell cycle progression and positivecontrol on cell differentiation.

In a preferred embodiment of the invention the cell cycle progressionrate is modified by downregulation of expression of a plant ICK proteinor a homologue or derivative thereof as defined in the presentinvention. ICK molecules interact with and inhibit the activity of cellcycle control molecules, e.g., Cdc2-related protein kinases or cyclins.Accordingly, decreased levels of ICK molecules result in a significantacceleration of the cell cycle progression. Downregulation of theexpression of ICK can promote and extend cell division activity in cellsthat normally become quiescent during the course of development and/oras a consequence of adverse growth conditions and/or as a consequence ofstress conditions. Downregulation of expression of ICK can, thus, beexpected to increase the frequency of the formation of lateral organsincluding leaves (resulting in increased bushiness), flowers (resultingin increased numbers of seeds or seed pods) and roots (resulting inincreased numbers of lateral roots). The timing of lateral organformation can also be altered, e.g., resulting in earlier flowering.Another expected effect of delaying cells to become quiescent is thedelayed occurrence of senescence. Downregulation of expression of ICK isfurthermore expected to enhance growth under conditions of, e.g., saltor drought stress.

When downregulation of expression of ICK is occurring at the whole plantlevel, an overall growth enhancing effect is to be expected, i.e.,recombinant plants will grow faster and/or will reach a larger size.This is particularly useful to increase the yield of, e.g., fodderplants, forage plants, leguminous plants, and wood-producing plants.Alternatively, downregulation of expression of ICK at the whole plantlevel may result in local growth enhancing effects, e.g., due to localand specific expression of the plant cellular proteins specificallytargeted by said ICK. When downregulation of expression of ICK isconfined to single cells, tissues, or organs of a plant, the growthenhancing effect will be confined to the single cells, tissues or organsof the plant. Particularly useful are restrictions of downregulation ofexpression of ICK to tissues or organs including seeds, fruits, tubers,roots, shoots, stems, and nodules to increase yield and/or size of thesetissues or organs.

Thus, a preferred embodiment of the present invention involvesdownregulation of expression of an ICK protein or a homologue orderivative thereof as defined herein, in a plant cell and/or tissueand/or organ to obtain enhanced growth and/or delayed senescence of theplant cell and/or tissue and/or organ, or to obtain enhanced formationof lateral organs from the plant tissue and/or organ.

In another preferred embodiment downregulation of expression of an ICKprotein or a homologue or derivative thereof as defined herein, in awhole plant results in enhanced growth and/or in increased frequency oflateral organ formation and/or delayed senescence of the plant.

Plant cells in which expression of an ICK is downregulated arefurthermore expected to be stimulated to go through endoreduplicationcycles, i.e., passage through consecutive cell cycles including DNAreplication but without intervening cytokinesis. Cells undergoingendoreduplication, thus, become polyploid.

Downregulation of expression of ICK can further be used to increase seedyield and/or seed size. Grain yield in crop plants is largely a functionof the amount of starch produced in the endosperm of the seed. Theamount of protein produced in the endosperm is also a contributingfactor to grain yield (Traas et al. (1998) Current Opin. Plant Biol. 1,498-503). In contrast, the embryo and aleurone layers contribute littlein terms of the total weight of the mature grain. By virtue of beinglinked to cell expansion and metabolic activity, endoreduplication isgenerally considered to be an important factor for increasing yield. Asgrain endosperm development initially includes extensiveendoreduplication (Olsen et al. (1999) Trends Plant Sci. 4, 253-257),enhancing, promoting or stimulating this process is likely to result inincreased grain yield. Enhancing, promoting or stimulating cell divisionduring seed development as described supra is an alternative way toincrease grain yield. In another aspect, the present invention alsofeatures a method for the production of SiO₂ from the peels or husks oflarger rice seeds. Methods for extraction and/or production of pure SiO₂from rice seed peels or husks are known in the art (e.g. Gorthy andPudukottah 1999) and units for production of SiO₂ from rice seed peelsare being set up (visit e.g.http://bisnis.doc.gov/bisnis/leads/990604sp.htm). SiO₂ has manyapplications including electronics, perfume industry and pharmacologyand silicone production.

Another embodiment of the current invention comprises cell cyclestage-specific, developmental stage-specific and/or tissue-specificdownregulation of expression of an ICK protein or a homologue orderivative thereof as defined herein. Downregulation of ICK expressioncan be obtained by using nucleotide sequences distinguishingalternatively polyadenylated transcripts of ICK (see Example 4). Thisapproach has potential advantages such as, e.g., the fact that aconstitutive promoter can be used to downregulate expression of ICKinstead of a cell cycle phase-specific, developmental stage-specific ortissue-specific promoter.

In yet another preferred embodiment of the invention the cell cycleprogression rate is significantly modified by ectopic expression of anICK protein or a homologue or derivative thereof as defined herein. AsICK molecules interact with and inhibit the activity of cell cyclecontrol, e.g., CDK, molecules, elevated levels of ICK result in asignificant inhibition of the cell cycle progression. Thus, effectsopposite to those obtainable as described for downregulation ofexpression of ICK can be expected. These opposite effects have usefulapplications as described infra. Ectopic expression of ICK at the wholeplant level can, e.g., create dwarfism. Ectopic expression of ICK inspecific cells, tissues or organs can be used to inhibit side shootformation in crops such as tomato. Ectopic expression of ICK may alsoconfer enhanced resistance to pathogens causing neoplastic plant growth,such as plant pathogenic bacteria including Agrobacterium tumefaciens,Rhodococcus fascians, Pseudomonas savastnoi, Xanthomonas campestris pvcitri and Erwinia herbicola, plant pathogenic fungi includingPlasmodiophora brassicae, Crinipellis perniciosa, Pucciniastrumgeoppertianum, Taphrina wiesneri, Ustilaga maydis, Exobasidium vaccinei,E. camelliae, Entorrhiza casparyana and Apiosporina morbosum.

Ectopic expression of an ICK molecule may also confer enhancedresistance or tolerance against pathogens which rely onendoreduplication events in the infected host cells to survive. Theectopic expression of ICK is expected to inhibit endoreduplicationevents. Pathogens relying on host cell endoreduplication to, forexample, establish a feeding structure, include nematodes such asHeterodera species and Meloidogyne species.

As used herein, the terms “ectopic expression” or “ectopicoverexpression” of a gene or a protein refer to expression patternsand/or expression levels of the gene or protein normally not occurringunder natural conditions.

By “cell fate and/or plant development and/or plant morphology and/orbiochemistry and/or physiology” is meant that one or more developmentaland/or morphological and/or biochemical and/or physiologicalcharacteristics of a plant is altered by the performance of one or moresteps pertaining to the invention described herein.

“Cell fate” includes the cell-type or cellular characteristics of aparticular cell that are produced during plant development or a cellularprocess therefor, in particular during the cell cycle or as aconsequence of a cell cycle process.

The term “plant development” or the term “plant developmentalcharacteristic” or similar terms shall, when used herein, be taken tomean any cellular process of a plant that is involved in determining thedevelopmental fate of a plant cell, in particular the specific tissue ororgan type into which a progenitor cell will develop. Cellular processesrelevant to plant development will be known to those skilled in the art.Such processes include, for example, morphogenesis, photomorphogenesis,shoot development, root development, vegetative development,reproductive development, stem elongation, flowering, and regulatorymechanisms involved in determining cell fate, in particular a process orregulatory process involving the cell cycle.

The term “plant morphology” or the term “plant morphologicalcharacteristic” or similar term will, when used herein, be understood bythose skilled in the art to include the external appearance of a plant,including any one or more structural features or combination ofstructural features thereof. Such structural features include the shape,size, number, position, color, texture, arrangement, and patternation ofany cell, tissue or organ or groups of cells, tissues or organs of aplant, including the root, stem, leaf, shoot, petiole, trichome, flower,petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm,seed coat, aleurone, fiber, fruit, cambium, wood, heartwood, parenchyma,aerenchyma, sieve element, phloem or vascular tissue.

The term “plant biochemistry” or the term “plant biochemicalcharacteristic” or similar term will, when used herein, be understood bythose skilled in the art to include the metabolic and catalyticprocesses of a plant, including primary and secondary metabolism and theproducts thereof, including any small molecules, macromolecules orchemical compounds, such as but not limited to starches, sugars,proteins, peptides, enzymes, hormones, growth factors, nucleic acidmolecules, celluloses, hemicelluloses, calloses, lectins, fibbers,pigments such as anthocyanins, vitamins, minerals, micronutrients, ormacronutrients, that are produced by plants.

The term “plant physiology” or the term “plant physiologicalcharacteristic” or similar term will, when used herein, be understood toinclude the functional processes of a plant, including developmentalprocesses such as growth, expansion and differentiation, sexualdevelopment, sexual reproduction, seed set, seed development, grainfilling, asexual reproduction, cell division, dormancy, germination,light adaptation, photosynthesis, leaf expansion, fibber production,secondary growth or wood production, amongst others; responses of aplant to externally-applied factors such as metals, chemicals, hormones,growth factors, environment and environmental stress factors (e.g.,anoxia, hypoxia, high temperature, low temperature, dehydration, light,day length, flooding, salt, heavy metals, amongst others), includingadaptive responses of plants to said externally-applied factors.

The ICK molecules of the present invention are useful in agriculture.The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used to modulate the protein levels or activityof a protein involved in the cell cycle, e.g., proteins involved in theG1/S and/or the G2/M transition in the cell cycle.

Thus, the ICK molecules of the present invention may be used tomodulate, e.g., enhance, crop yields; modulate, e.g., attenuate, stress,e.g., heat or nutrient deprivation, tolerance; modulate tolerance topests and diseases; modulate plant architecture; modulate plant qualitytraits; or modulate plant reproduction and seed development.

The ICK molecules of the present invention may also be used to modulateendoreduplication in storage cells, storage tissues, and/or storageorgans of plants or parts thereof. The term “endoreduplication” includesrecurrent DNA replication without consequent mitosis and cytokinesis.Preferred target storage organs and parts thereof for the modulation ofendoreduplication are, for example, seeds (such as from cereals, oilseedcrops), roots (such as in sugar beet), tubers (such as in potatoes) andfruits (such as in vegetables and fruit species). Increasedendoreduplication in storage organs, and parts thereof, correlates withenhanced storage capacity and, thus, with improved yield. In anotherembodiment of the invention, the endoreduplication of a whole plant ismodulated.

B. Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to ICK proteins, have a stimulatory or inhibitory effect on,for example, ICK expression or ICK activity, or have a stimulatory orinhibitory effect on, for example, the expression or activity of an ICKsubstrate.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of an ICK protein or polypeptideor biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of an ICK protein or polypeptideor biologically active portion thereof, e.g., modulate the ability ofICK to interact with its cognate ligand. The test compounds of thepresent invention can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra).

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing an ICK target molecule (e.g., a plantcyclin dependent kinase) with a test compound and determining theability of the test compound to modulate (e.g. stimulate or inhibit) theactivity of the ICK target molecule. Determining the ability of the testcompound to modulate the activity of an ICK target molecule can beaccomplished, for example, by determining the ability of the ICK proteinto bind to or interact with the ICK target molecule, or by determiningthe ability of the target molecule, e.g., the plant cyclin dependentkinase, to phosphorylate a protein.

The ability of the target molecule, e.g., the plant cyclin dependentkinase, to phosphorylate a protein can be determined by, for example, anin vitro kinase assay. Briefly, a protein can be incubated with thetarget molecule, e.g., the plant cyclin dependent kinase, andradioactive ATP, e.g., [γ-³²P] ATP, in a buffer containing MgCl₂ andMnCl₂, e.g., 10 mM MgCl₂ and 5 mM MnCl₂. Following the incubation, theimmunoprecipitated protein can be separated by SDS-polyacrylamide gelelectrophoresis under reducing conditions, transferred to a membrane,e.g., a PVDF membrane, and autoradiographed. The appearance ofdetectable bands on the autoradiograph indicates that the protein hasbeen phosphorylated. Phosphoaminoacid analysis of the phosphorylatedsubstrate can also be performed in order to determine which residues onthe protein are phosphorylated. Briefly, the radiophosphorylated proteinband can be excised from the SDS gel and subjected to partial acidhydrolysis. The products can then be separated by one-dimensionalelectrophoresis and analyzed on, for example, a phosphoimager andcompared to ninhydrin-stained phosphoaminoacid standards.

Determining the ability of the ICK protein to bind to or interact withan ICK target molecule can be accomplished by determining directbinding. Determining the ability of the ICK protein to bind to orinteract with an ICK target molecule can be accomplished, for example,by coupling the ICK protein with a radioisotope or enzymatic label suchthat binding of the ICK protein to an ICK target molecule can bedetermined by detecting the labeled ICK protein in a complex. Forexample, ICK molecules, e.g., ICK proteins, can be labeled with ¹²⁵I,³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotopedetected by direct counting of radioemmission or by scintillationcounting. Alternatively, ICK molecules can be enzymatically labeledwith, for example, horseradish peroxidase, alkaline phosphatase, orluciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound to modulate the interaction between ICK and its targetmolecule, without the labeling of any of the interactants. For example,a microphysiometer can be used to detect the interaction of ICK with itstarget molecule without the labeling of either ICK or the targetmolecule. McConnell, H. M. et al. (1992) Science 257:1906-1912. As usedherein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between compound and receptor.

In a preferred embodiment, determining the ability of the ICK protein tobind to or interact with an ICK target molecule can be accomplished bydetermining the activity of the target molecule. For example, theactivity of the target molecule can be determined by detecting inductionof a cellular second messenger of the target, detectingcatalytic/enzymatic activity of the target an appropriate substrate,detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., chloramphenicol acetyltransferase), or detecting a target-regulated cellular response.

In yet another embodiment, an assay of the present invention is acell-free assay in which an ICK protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to the ICK protein or biologically active portionthereof is determined. Binding of the test compound to the ICK proteincan be determined either directly or indirectly as described above. In apreferred embodiment, the assay includes contacting the ICK protein orbiologically active portion thereof with a known compound which bindsICK to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith an ICK protein, wherein determining the ability of the testcompound to interact with an ICK protein comprises determining theability of the test compound to preferentially bind to ICK orbiologically active portion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which an ICKprotein or biologically active portion thereof is contacted with a testcompound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the ICK protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of an ICK protein can beaccomplished, for example, by determining the ability of the ICK proteinto bind to an ICK target molecule by one of the methods described abovefor determining direct binding. Determining the ability of the ICKprotein to bind to an ICK target molecule can also be accomplished usinga technology such as real-time Biomolecular Interaction Analysis (BIA).Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 andSzabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein,“BIA” is a technology for studying biospecific interactions in realtime, without labeling any of the interactants (e.g., BIAcore). Changesin the optical phenomenon of surface plasmon resonance (SPR) can be usedas an indication of real-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of an ICK protein can be accomplishedby determining the ability of the ICK protein to further modulate theactivity of an ICK target molecule (e.g., an ICK mediated signaltransduction pathway component). For example, the activity of theeffector molecule on an appropriate target can be determined, or thebinding of the effector to an appropriate target can be determined aspreviously described.

In yet another embodiment, the cell-free assay involves contacting anICK protein or biologically active portion thereof with a known compoundwhich binds the ICK protein to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with the ICK protein, wherein determining theability of the test compound to interact with the ICK protein comprisesdetermining the ability of the ICK protein to preferentially bind to ormodulate the activity of an ICK target molecule.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of proteins (e.g., ICK proteinsor biologically active portions thereof). In the case of cell-freeassays in which a membrane-bound form a protein is used it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of the protein is maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either ICK or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to an ICK protein, or interaction ofan ICK protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/ICK fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or ICK protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of ICKbinding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either an ICKprotein or an ICK target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated ICK protein ortarget molecules can be prepared from biotin-NHS(N-hydroxy-succinimide)using techniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with ICK protein or target molecules but which donot interfere with binding of the ICK protein to its target molecule canbe derivatized to the wells of the plate, and unbound target or ICKprotein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the ICK protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the ICK protein or target molecule.

In another embodiment, modulators of ICK expression are identified in amethod wherein a cell is contacted with a candidate compound and theexpression of ICK mRNA or protein in the cell is determined. The levelof expression of ICK mRNA or protein in the presence of the candidatecompound is compared to the level of expression of ICK mRNA or proteinin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of ICK expression based on thiscomparison. For example, when expression of ICK mRNA or protein isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of ICK mRNA or protein expression.Alternatively, when expression of ICK mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of ICK mRNA or protein expression. The level of ICK mRNA orprotein expression in the cells can be determined by methods describedherein for detecting ICK mRNA or protein.

In yet another aspect of the invention, the ICK proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with ICK (“ICK-binding proteins” or “ICK-bp”) and are involvedin ICK activity. Such ICK-binding proteins are also likely to beinvolved in the propagation of signals by the ICK proteins or ICKtargets as, for example, downstream elements of an ICK-mediatedsignaling pathway. Alternatively, such ICK-binding proteins are likelyto be ICK inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for an ICK protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming an ICK-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the ICKprotein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate plant or animal model. For example, an agent identifiedas described herein (e.g., an ICK modulating agent, an antisense ICKnucleic acid molecule, an ICK-specific antibody, or an ICK-bindingpartner) can be used in a plant or animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in aplant or animal model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for the agriculturaland therapeutic uses described herein.

C. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; identify an individual from aminute biological sample (tissue typing); and aid in forensicidentification of a biological sample. Once the sequence (or a portionof the sequence) of a gene has been isolated, this sequence can be usedto map the location of the gene on a chromosome. This process is calledchromosome mapping. Accordingly, portions or fragments of the ICKnucleotide sequences, described herein, can be used to map the locationof the ICK genes on a chromosome. The mapping of the ICK sequences tochromosomes is an important first step in correlating these sequenceswith genes associated with disease.

Briefly, ICK genes can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp in length) from the ICK nucleotide sequences.Computer analysis of the ICK sequences can be used to predict primersthat do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers can then be usedfor PCR screening of cell hybrids containing individual plant or humanchromosomes. Only those hybrids containing the plant or human genecorresponding to the ICK sequences will yield an amplified fragment.

Other mapping strategies which can similarly be used to map an ICKsequence to its chromosome include in situ hybridization (described inFan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between plants affected andunaffected with a disease associated with the ICK gene, can bedetermined. If a mutation is observed in some or all of the affectedplants but not in any unaffected plants, then the mutation is likely tobe the causative agent of the particular disease. Comparison of affectedand unaffected plants generally involves first looking for structuralalterations in the chromosomes, such as deletions or translocations thatare visible from chromosome spreads or detectable using PCR based onthat DNA sequence. Ultimately, complete sequencing of genes from severalplants can be performed to confirm the presence of a mutation and todistinguish mutations from polymorphisms.

II. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode ICK proteins or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify ICK-encoding nucleic acids (e.g., ICK mRNA) andfragments for use as PCR primers for the amplification or mutation ofICK nucleic acid molecules. As used herein, the term “nucleic acidmolecule” is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA), chimeric RNA/DNA oligonucleotides,and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. For example, with regards to genomic DNA, the term“isolated” includes nucleic acid molecules which are separated from thechromosome with which the genomic DNA is naturally associated.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated ICK nucleic acid molecule can contain less than about 5 kb,4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, 9, 43, 45, 54 or 56, or a portion thereof, can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. For example, using all or portion of the nucleic acidsequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56, as ahybridization probe, ICK nucleic acid molecules can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56 can be isolated by thepolymerase chain reaction (PCR) using synthetic oligonucleotide primersdesigned based upon the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 43, 45, 54 or 56, respectively.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to ICK nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO: 1, 2, 3,4, 5, 6, 7, 8, 9, 43, 45, 54 or 56.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9,43, 45, 54 or 56, or a portion of any of these nucleotide sequences. Anucleic acid molecule which is complementary to the nucleotide sequenceshown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56, is onewhich is sufficiently complementary to the nucleotide sequence shown inSEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56, respectively,such that it can hybridize to the nucleotide sequence shown in SEQ IDNO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56, respectively, therebyforming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% ormore identical to the nucleotide sequence (e.g., to the entire length ofthe nucleotide sequence) shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9,43, 45, 54 or 56, or a portion of any of these nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7,8, 9, 43, 45, 54 or 56, for example a fragment which can be used as aprobe or primer or a fragment encoding a biologically active portion ofan ICK protein. The nucleotide sequence determined from the cloning ofthe ICK gene allows for the generation of probes and primers designedfor use in identifying and/or cloning other ICK family members, as wellas ICK homologues from other species. The probe/primer typicallycomprises substantially purified oligonucleotide. The oligonucleotidetypically comprises a region of nucleotide sequence that hybridizesunder stringent conditions to at least about 12 or 15, preferably about20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense sequence of SEQ ID NO:1, 2, 3, 4, 5,6, 7, 8, 9, 43, 45, 54 or 56, or of a naturally occurring allelicvariant or mutant of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or56. In an exemplary embodiment, a nucleic acid molecule of the presentinvention comprises a nucleotide sequence which is at least 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800nucleotides in length and hybridizes under stringent hybridizationconditions to a nucleic acid molecule of SEQ ID NO:1, 2, 3, 4, 5, 6, 7,8, 9, 43, 45, 54 or 56.

Probes based on the ICK nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissues which misexpress an ICK protein, such as by measuring a level ofan ICK-encoding nucleic acid in a sample of cells from a subject e.g.,detecting ICK mRNA levels or determining whether a genomic ICK gene hasbeen mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of anICK protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56, whichencodes a polypeptide having an ICK biological activity (the biologicalactivities of the ICK proteins are described herein), expressing theencoded portion of the ICK protein (e.g., by recombinant expression invitro) and assessing the activity of the encoded portion of the ICKprotein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 43, 45, 54 or 56, due to the degeneracy of the genetic code and,thus, encode the same ICK proteins as those encoded by the nucleotidesequence shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56.In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding an ICK protein.

In addition to the ICK nucleotide sequences shown in SEQ ID NO: 1, 2, 3,4, 5, 6, 7, 8, 9, 43, 45, 54 or 56, it will be appreciated by thoseskilled in the art that DNA sequence polymorphisms that lead to changesin the amino acid sequences of the ICK proteins may exist within apopulation (e.g., a rice plant population). Such genetic polymorphism inthe ICK genes may exist among individuals within a population due tonatural allelic variation. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules which include an openreading frame encoding an ICK protein, preferably a plant ICK protein,and can further include non-coding regulatory sequences, and introns.Such natural allelic variations include both functional andnon-functional ICK proteins and can typically result in 1-5% variance inthe nucleotide sequence of an ICK gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms in ICK genes that arethe result of natural allelic variation and that do not alter thefunctional activity of an ICK protein are intended to be within thescope of the invention.

Natural allelic variants are further include molecules that comprisesingle nucleotide polymorphisms (SNPs) as well as smallinsertion/deletion polymorphisms (INDELs; the size of INDELs is usuallyless than about 100 bp). SNPs and INDELs form the largest set ofsequence variants in naturally occurring polymorphic strains of mostorganisms. They are helpful in mapping genes and in discovery of genesand gene functions. They are furthermore helpful in the identificationof genetic loci, e.g., plant genes, involved in determining processessuch as growth rate, plant size and plant yield, plant vigor, diseaseresistance, stress tolerance and the like. Many techniques are nowadaysavailable to identify SNPs and/or INDELs including (i) PCR followed bydenaturing high performance liquid chromatography (DHPLC; e.g., Cho etal. (1999) Nature Genet 23, 203-207); (ii) constant denaturant capillaryelectrophoresis (CDCE) combined with high-fidelity PCR (e.g.,Li-Sucholeiki et al. (1999) Electrophoresis 20, 1224-1232); (iii)denaturing gradient gel electrophoresis (Fischer and Lerman (1983) Proc.Natl. Acad. Sci. USA 80, 1579-1583); (iv) matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS;e.g., Ross et al (2000) Biotechniques 29, 620-629); (v) real-timefluorescence monitoring PCR assays (Tapp et al (2000) Biotechniques 28,732-738); (vi) Acrydite™ gel technology (Kenney et al (1998)Biotechniques 25; 516-521); (vii) cycle dideoxy fingerprinting (CddF;Langemeier et al (1994) Biotechniques 17, 484-490); (vii) single-strandconformation polymorphism (SSCP) analysis (Vidal-Puig and Moller (1994)Biotechniques 17, 490-496) and (ix) mini-sequencing primer extensionreaction (Syvanen (1999) Hum Mutat 13, 1-10). The technique of‘Targeting Induced Local Lesions in Genomes’ (TILLING; McCallum et al.(2000) Nat. Biotechnol 8, 455-457; Plant Physiol 123, 439-442), which isa variant of (i) supra, can also be applied to rapidly identify analtered gene in, e.g., chemically mutagenized plant individuals showinginteresting phenotypes.

Differences in preferred codon usage are illustrated below forAgrobacterium tumefaciens (a bacterium), A. thaliana, M. sativa (twodicotyledonous plants) and Oryza sativa (a monocotyledonous plant). Forexample, the codon GGC (for glycine) is the most frequently used codonin A. tumefaciens (36.2‰), is the second most frequently used codon inO. sativa but is used at much lower frequencies in A. thaliana and M.sativa (9‰ and 8.4‰, respectively). Of the four possible codons encodingglycine the GGC codon is most preferably used in A. tumefaciens and O.sativa. However, in A. thaliana the GGA (and GGU) codon is mostpreferably used, whereas in M. sativa the GGU (and GGA) codon is mostpreferably used.

Moreover, nucleic acid molecules encoding other ICK family members and,thus, which have a nucleotide sequence which differs from the ICKsequences of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56 areintended to be within the scope of the invention. For example, anotherICK cDNA can be identified based on the nucleotide sequence of the plantICK molecules described herein. Moreover, nucleic acid moleculesencoding ICK proteins from different species and, thus, which have anucleotide sequence which differs from the ICK sequences of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56 are intended to be withinthe scope of the invention. For example, a corn ICK cDNA can beidentified based on the nucleotide sequence of a rice ICK.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the ICK cDNAs of the invention can be isolated based ontheir homology to the ICK nucleic acids disclosed herein using the cDNAsdisclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8,9, 43, 45, 54 or 56. In other embodiment, the nucleic acid is at least30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600nucleotides in length. As used herein, the term “hybridizes understringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 30%,40%, 50%, or 60% identical to each other typically remain hybridized toeach other. Preferably, the conditions are such that sequences at leastabout 70%, more preferably at least about 80%, even more preferably atleast about 85% or 90% identical to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred,non-limiting example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C.,preferably at 55° C., more preferably at 60° C., and even morepreferably at 65° C. Ranges intermediate to the above-recited values,e.g., at 60-65° C. or at 55-60° C. are also intended to be encompassedby the present invention. Preferably, an isolated nucleic acid moleculeof the invention that hybridizes under stringent conditions to thesequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56corresponds to a naturally-occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the ICK sequencesthat may exist in nature, the skilled artisan will further appreciatethat changes can be introduced by mutation into the nucleotide sequencesof SEQ ID NO:1, 2, 3, 4, 5, 6, 7; 8, 9, 43, 45, 54 or 56, therebyleading to changes in the amino acid sequence of the encoded ICKproteins, without altering the functional ability of the ICK proteins.For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in thesequence of an ICK protein. A “non-essential” amino acid residue is aresidue that can be altered from the wild-type sequence of ICK withoutaltering the biological activity, whereas an “essential” amino acidresidue is required for biological activity. For example, amino acidresidues that are conserved among the ICK proteins of the presentinvention, are predicted to be particularly unamenable to alteration.Furthermore, additional amino acid residues that are conserved betweenthe ICK proteins of the present invention and other ICK family membersare not likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding ICK proteins that contain changes in amino acidresidues that are not essential for activity.

An isolated nucleic acid molecule encoding an ICK protein homologous tothe ICK proteins of the present invention can be created by introducingone or more nucleotide substitutions, additions or deletions into thenucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54or 56, such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced into SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in an ICKprotein is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of an ICK coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for ICK biological activity to identify mutants that retainactivity. Following mutagenesis of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,43, 45, 54 or 56, the encoded protein can be expressed recombinantly andthe activity of the protein can be determined. Another alternativeembodiment comprises targeted in vivo gene correction or modificationwhich can be achieved by chimeric RNA/DNA oligonucleotides (e.g. Yoon etal. (1996), Proc. Natl. Acad. Sci. USA 93, 2071-2076).

In a preferred embodiment, a mutant ICK protein can be assayed for theability to: (1) inhibit CDK activity (e.g., cyclin-CDK activity); (2)regulate cyclin-CDK complex assembly; (3) regulate the commitment ofcells to divide, e.g., by integrating mitogenic and antimitogenicsignals; (4) regulate cell cycle progression; (5) regulate DNAreplication and/or DNA repair; (6) regulate gene transcription; (7)regulate cyclin degradation; (8) modulate cell cycle withdrawal and/orcell differentiation; (9) regulate cell death, e.g., apoptosis; (10)control organ (e.g., plant organ) and/or organism (e.g., plant organism)size; and (11) regulate endoreduplication.

In addition to the nucleic acid molecules encoding ICK proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire ICK coding strand, or only to a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding ICK. Theterm “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues. Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding ICK. The term “noncoding region” refers to 5′ and 3′ sequenceswhich flank the coding region that are not translated into amino acids(i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding ICK disclosed herein,antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of ICK mRNA,but more preferably is an oligonucleotide which is antisense to only aportion of the coding or noncoding region of ICK mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of ICK mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection). Preferably, production of antisense nucleicacids in plants occurs by means of a stably integrated transgenecomprising a promoter operative in plants, an antisense oligonucleotide,and a terminator.

Other known nucleotide modifications include methylation, cyclizationand ‘caps’ and substitution of one or more of the naturally occurringnucleotides with an analog such as inosine. Modifications of nucleotidesinclude the addition of acridine, amine, biotin, cascade blue,cholesterol, Cy3®, Cy5®, Cy5.5® Dabcyl, digoxigenin, dinitrophenyl,Edans, 6-FAM, fluorescein, 3′-glyceryl, HEX, IRD-700, IRD-800, JOE,phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET,AMCA-S®, SE, BODIPY®, Marina Blue®, Pacific Blue®, Oregon Green®,Rhodamine Green®, Rhodamine Red®, Rhodol Green® and Texas Red®.Polynucleotide backbone modifications include methylphosphonate,2′-OMe-methylphosphonate RNA, phosphorothiorate, RNA, 2′-OMeRNA. Basemodifications include 2-amino-dA, 2-aminopurine, 3′-(ddA), 3′dA(cordycepin), 7-deaza-dA, 8-Br-dA, 8-oxo-dA, N⁶-Me-dA, abasic site(dSpacer), biotin dT, 2′-OMe-5Me-C, 2′-OMe-propynyl-C, 3′-(5-Me-dC),3′-(ddC), 5-Br-dC, 5-I-dC, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA,convertible dC, convertible dG, convertible dT, convertible dU,7-deaza-dG, 8-Br-dG, 8-oxo-dG, O⁶-Me-dG, S6-DNP-dG, 4-methyl-indole,5-nitroindole, 2′-OMe-inosine, 2′-dI, 0⁶-phenyl-dI, 4-methyl-indole,2′-deoxynebularine, 5-nitroindole, 2-aminopurine, dP (purine analogue),dK (pyrimidine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT,biotin-dT, carboxy-dT, O⁴-Me-dT, O⁴-triazol dT, 2′-OMe-propynyl-U,5-Br-dU, 2′-dU, 5-F-dU, 5-I-dU, O⁴-triazol dU.

The antisense nucleic acid molecules of the invention are typicallyintroduced into a plant or administered to a subject or generated insitu such that they hybridize with or bind to cellular mRNA and/orgenomic DNA encoding an ICK protein to thereby inhibit expression of theprotein, e.g., by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarity to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule which binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An example of a route ofintroduction or administration of antisense nucleic acid molecules ofthe invention include transformation in a plant or direct injection at atissue site in a subject. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, antisensemolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface, e.g., by linking theantisense nucleic acid molecules to peptides or antibodies which bind tocell surface receptors or antigens. The antisense nucleic acid moleculescan also be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a constitutive promoter or astrong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330). In another embodiment, the antisense nucleic acidmolecule further comprises a sense nucleic acid molecule complementaryto the antisense nucleic acid molecule. Gene silencing methods based onsuch nucleic acid molecules are well known to the skilled artisan (e.g.,Grierson et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO99/53050).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveICK mRNA transcripts to thereby inhibit translation of ICK mRNA. Aribozyme having specificity for an ICK-encoding nucleic acid can bedesigned based upon the nucleotide sequence of an ICK cDNA disclosedherein (i.e., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 43, 45, 54 or 56).For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in anICK-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; andCech et al. U.S. Pat. No. 5,116,742. Alternatively, ICK mRNA can be usedto select a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993)Science 261:1411-1418. The use of ribozymes for gene silencing in plantsis known in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne etal. (1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen etal. (1997) WO 97/13865 and Scott et al. (1997) WO/97/38116).

Alternatively, ICK gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the ICK(e.g., the ICK promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the ICK gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. NY. Acad. Sci. 660:27-36; and Maher, L. J.(1992) Bioassays 14(12):807-15.

In yet another embodiment, the ICK nucleic acid molecules of the presentinvention can be modified at the base moiety, sugar moiety or phosphatebackbone to improve, e.g., the stability, hybridization, or solubilityof the molecule. For example, the deoxyribose phosphate backbone of thenucleic acid molecules can be modified to generate peptide nucleic acids(see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1):5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” referto nucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc.Natl. Acad. Sci. 93: 14670-675.

PNAs of ICK nucleic acid molecules can be used for increasing crop yieldin plants or in therapeutic and diagnostic applications. For example,PNAs can be used as antisense or antigene agents for sequence-specificmodulation of gene expression by, for example, inducing transcription ortranslation arrest or inhibiting replication. PNAs of ICK nucleic acidmolecules can also be used in the analysis of single base pair mutationsin a gene, (e.g., by PNA-directed PCR clamping); as ‘artificialrestriction enzymes’ when used in combination with other enzymes, (e.g.,S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNAsequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefesupra).

In another embodiment, PNAs of ICK can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of ICK nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup B. (1996) supra and Finn P. J. et al.(1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain canbe synthesized on a solid support using standard phosphoramiditecoupling chemistry and modified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization triggered cleavage agents (See, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

III. Isolated ICK Proteins and Anti-ICK Antibodies

One aspect of the invention pertains to isolated ICK proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-ICK antibodies. In oneembodiment, native ICK proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, ICK proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, an ICK protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the ICKprotein is derived, or substantially free from chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of ICK protein in whichthe protein is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof ICK protein having less than about 30% (by dry weight) of non-ICKprotein (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-ICK protein, still more preferablyless than about 10% of non-ICK protein, and most preferably less thanabout 5% non-ICK protein. When the ICK protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of ICK protein in which the protein isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of ICK protein having less than about 30% (by dry weight)of chemical precursors or non-ICK chemicals, more preferably less thanabout 20% chemical precursors or non-ICK chemicals, still morepreferably less than about 10% chemical precursors or non-ICK chemicals,and most preferably less than about 5% chemical precursors or non-ICKchemicals.

Biologically active portions of an ICK protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the ICK protein, which include lessamino acids than the full length ICK proteins, and exhibit at least oneactivity of an ICK protein. Typically, biologically active portionscomprise a domain or motif with at least one activity of the ICKprotein. A biologically active portion of an ICK protein can be apolypeptide which is, for example, at least 10, 25, 50, 100 or moreamino acids in length.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the GAP program in the GCGsoftware package (available at http://www.gcg.com), using either aBlosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10,8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (available at http://www.gcg.com), using a NWSgapdna.CMP matrixand a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to ICK nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to ICK proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The invention also provides ICK chimeric or fusion proteins. As usedherein, an ICK “chimeric protein” or “fusion protein” comprises an ICKpolypeptide operatively linked to a non-ICK polypeptide. An “ICKpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to ICK, whereas a “non-ICK polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the ICK protein, e.g., aprotein which is different from the ICK protein and which is derivedfrom the same or a different organism. The non-ICK polypeptide can, forexample, be (histidine)₆-tag, glutathione S-transferase, protein A,maltose-binding protein, dihydrofolate reductase, Tag.100 epitope(EETARFQPGYRS; SEQ ID NO:37), c-myc epitope (EQKLISEEDL; SEQ ID NO:38),FLAG®-epitope (DYKDDDK; SEQ ID NO:39), lacZ, CMP (calnodulin-bindingpeptide), HA epitope (YPYDVPDYA; SEQ ID NO:40), protein C epitope(EDQVDPRLIDGK; SEQ ID NO:41) or VSV epitope (YTDIEMNRLGK; SEQ ID NO:42).

Within an ICK fusion protein the ICK polypeptide can correspond to allor a portion of an ICK protein. In a preferred embodiment, an ICK fusionprotein comprises at least one biologically active portion of an ICKprotein. In another preferred embodiment, an ICK fusion proteincomprises at least two biologically active portions of an ICK protein.Within the fusion protein, the term “operatively linked” is intended toindicate that the ICK polypeptide and the non-ICK polypeptide are fusedin-frame to each other. The non-ICK polypeptide can be fused to theN-terminus or C-terminus of the ICK polypeptide.

For example, in one embodiment, the fusion protein is a GST-ICK fusionprotein in which the ICK sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant ICK.

In another embodiment, the fusion protein is an ICK protein containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., plant or mammalian host cells), expression and/or secretion ofICK can be increased through use of a heterologous signal sequence.

The ICK fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a plant or a subject invivo. The ICK fusion proteins can be used to affect the bioavailabilityof an ICK substrate. Use of ICK fusion proteins may be usefulagriculturally for the increase of crop yields or therapeutically forthe treatment of cellular growth related disorders, e.g., cancer.Moreover, the ICK-fusion proteins of the invention can be used asimmunogens to produce anti-ICK antibodies in a subject, to purify ICKligands and in screening assays to identify molecules which modulate theinteraction of ICK with an ICK substrate, e.g., a CDK molecule or acyclin molecule.

Preferably, an ICK chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A ICK-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the ICK protein.

The present invention also pertains to variants of the ICK proteinswhich function as either ICK agonists (mimetics) or as ICK antagonists.Variants of the ICK proteins can be generated by mutagenesis, e.g.,discrete point mutation or truncation of an ICK protein. An agonist ofthe ICK proteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of an ICK protein.An antagonist of an ICK protein can inhibit one or more of theactivities of the naturally occurring form of the ICK protein by, forexample, competitively modulating a cellular activity of an ICK protein.Thus, specific biological effects can be elicited by treatment with avariant of limited function. In one embodiment, treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of theICK protein.

In one embodiment, variants of an ICK protein which function as eitherICK agonists (mimetics) or as ICK antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of an ICK protein for ICK protein agonist or antagonist activity. In oneembodiment, a variegated library of ICK variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of ICK variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential ICK sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of ICK sequences therein. There are avariety of methods which can be used to produce libraries of potentialICK variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential ICK sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477.

In addition, libraries of fragments of an ICK protein coding sequencecan be used to generate a variegated population of ICK fragments forscreening and subsequent selection of variants of an ICK protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of an ICK coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the ICK protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of ICK proteins. The mostwidely used techniques, which are amenable to high throughput analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify ICK variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated ICK library. For example, a library of expression vectors canbe transfected into a cell line which ordinarily synthesizes andsecretes ICK. The transfected cells are then cultured such that ICK anda particular mutant ICK are secreted and the effect of expression of themutant on ICK activity in cell supernatants can be detected, e.g., byany of a number of enzymatic assays. Plasmid DNA can then be recoveredfrom the cells which score for inhibition, or alternatively,potentiation of ICK activity, and the individual clones furthercharacterized.

An isolated ICK protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind ICK using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length ICK protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of ICK for use as immunogens. Theantigenic peptide of ICK comprises at least 8 amino acid residues andencompasses an epitope of ICK such that an antibody raised against thepeptide forms a specific immune complex with ICK. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

Preferred epitopes encompassed by the antigenic peptide are regions ofICK that are located on the surface of the protein, e.g., hydrophilicregions.

An ICK immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed ICK protein or a chemically synthesizedICK polypeptide. The preparation can further include an adjuvant, suchas Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic ICK preparation induces a polyclonal anti-ICK antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-ICKantibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as ICK. Examplesof immunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind ICK. The term “monoclonalantibody” or “monoclonal antibody composition”, as used herein, refersto a population of antibody molecules that contain only one species ofan antigen binding site capable of immunoreacting with a particularepitope of ICK A monoclonal antibody composition thus typically displaysa single binding affinity for a particular ICK protein with which itimmunoreacts.

Polyclonal anti-ICK antibodies can be prepared as described above byimmunizing a suitable subject with an ICK immunogen. The anti-ICKantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized ICK. If desired, the antibody moleculesdirected against ICK can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-ICK antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique(Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med, 54:387-402; M. L. Gefter et al. (1977) SomaticCell Genet. 3:231-36). Briefly, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with an ICK immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds ICK.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-ICK monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, YaleJ. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindICK, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-ICK antibody can be identified and isolated by screeninga recombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with ICK to thereby isolate immunoglobulinlibrary members that bind ICK. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J.Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gramet al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al.(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. AcidRes. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-ICK antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-ICK antibody (e.g., monoclonal antibody) can be used to isolateICK by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-ICK antibody can facilitate thepurification of natural ICK from cells and of recombinantly produced ICKexpressed in host cells. Moreover, an anti-ICK antibody can be used todetect ICK protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of the ICKprotein. These antibodies can also be used, for example, for theimmunoprecipitation and immunolocalization of proteins according to theinvention as well as for the monitoring of the synthesis of suchproteins, for example, in recombinant organisms, and for theidentification of compounds interacting with the protein according tothe invention.

Anti-ICK antibodies can be used diagnostically to monitor protein levelsin tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen. Detectioncan be facilitated by coupling (i.e., physically linking) the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, -galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

IV. Computer Readable Means

The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of mediums to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequences of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORI's)) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exist in nature or in purified form.

In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein “computer readable media” includes any medium thatcan be read and accessed directly by a computer. Such media include, butare not limited to: magnetic storage media, such as floppy discs, harddisc storage medium, and magnetic tape; optical storage media such aCD-ROM; electrical storage media such as RAM and ROM; and hybrids ofthese categories such as magnetic/optical storage media. The skilledartisan will readily appreciate how any of the presently known computerreadable mediums can be used to create a manufacture comprising computerreadable medium having recorded thereon a nucleotide or amino acidsequence of the present invention.

As used herein “recorded” refers to a process of storing information oncomputer readable medium. The skilled artisan can readily adopt any ofthe presently known methods for recording information on a computerreadable medium to generate manufactures comprising the nucleotide oramino acid sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase Oracle, orthe like. The skilled artisan can readily adapt any number ofdataprocessor structuring formats (e.g., text file or database) in orderto obtain computer readable medium having recorded thereon thenucleotide sequence information of the present invention.

By providing the nucleotide or amino acid sequences of the invention incomputer readable form, the skilled artisan can routinely access thesequence information for a variety of purposes. For example, one skilledin the art can use the nucleotide or amino acid sequences of theinvention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identity fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotide or two or more amino acids. A skilledartisan can readily recognize that the longer a target sequence is, theless likely a target sequence will be present as a random occurrence inthe database. The most preferred sequence length of a target sequence isfrom about 10 to 100 amino acids or form about 30 to 300 nucleotideresidues. However, it is well recognized that commercially importantfragments, such as sequence fragments involved in gene expression andprotein processing, may be shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knownalgorithms are disclosed publicly and a variety of commerciallyavailable software of conducting search means are and can be used in thecomputer-based systems of the present invention. Examples of suchsoftware include, but are not limited to, MacPatter (FMBL), BLASTN andBASIX (NCBIA).

For example, software which implements the BLAST (Altschul et al. (1990)J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp. Chem.17:203-207) search algorithms on a Sybase system can be used to identifyopen reading frames (ORFs) of the sequences of the invention whichcontain homology to ORFs or proteins from other libraries. Such ORFs areprotein encoding fragments and are useful in producing commerciallyimportant proteins such as enzyme used in various reactions and in theproduction of commercially useful metabolites.

Furthermore, folding simulations and computer redesign of structuralmotifs of the protein of the invention can be performed usingappropriate computer programs (Olszewski et al. 1996, Hoffman et al.1995). Computer modeling of protein folding can be used for theconformational and energetic analysis of detailed peptide and proteinmodels (Monge et al. 1995, Renouf and Hounsell 1995). In particular, theappropriate programs can be used for the identification of interactivesites of the ICK and cyclin-dependent kinases, its ligand or otherinteracting proteins by computer assisted searches for complementarypeptide sequences (Fassina and Melli 1994). Further appropriate computersystems for the design of protein and peptides are described in theprior art, for example in Berry and Brenner (1994), Wodak (1987). Paboand Suchanek (1986). The results obtained form the above-describedcomputer analysis can be used for, e.g. the preparation ofpeptidomimetics of the proteins of the invention or fragments thereof.

Furthermore, a three-dimensional and/or crystallographic structure ofthe protein of the invention can be used for the design ofpeptidomimetic inhibitors of the biological activity of the protein ofthe invention (Rose et al. 1996, Rutenber et al. 1996).

V. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an ICK protein(or a portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, e.g., a plant cell, which means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression, whichis operatively linked to the nucleic acid sequence to be expressed.Within a recombinant expression vector, “operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cell and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, and the like. The expression vectors of the invention can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein (e.g., ICK proteins, mutant forms of ICK proteins,fusion proteins, and the like).

The vectors of the invention comprise a selectable and/or scorablemarker. Selectable marker genes useful for the selection of transformedplant cells, callus, plant tissue and plants are well known to thoseskilled in the art and comprise, for example, antimetabolite resistanceas the basis of selection for dhfr, which confers resistance tomethotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994),143-149); npt, which confers resistance to the aminoglycosides neomycin,kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995)and hygro, which confers resistance to hygromycin (Marsh, Gene 32(1984), 481-485). Additional selectable genes have been described,namely trpB, which allow cells to utilize indole in place of tryptophan;hisD, which allows cells to utilize histinol in place of histidine(Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047);mannose-6-phosphate isomerase which allows cells to utilize mannose (WO94/20627) and ODC (omithine decarboxylase) which confers resistance tothe ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine,DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology,Cold Spring Harbor Laboratory ed.) or deaminase from Aspergillus terreuswhich confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol.Biochem. 59 (1995), 2336-2338).

Useful scorable markers are also known to those skilled in the art andare commercially available. Advantageously, the marker is a geneencoding luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J.Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett.389 (1996), 4447) or β-glucuronidase (Jefferson, EMBO J. 6 (1987),3901-3907). This embodiment is particularly useful for simple and rapidscreening of cells, tissues and organisms containing a vector of theinvention.

A “plant promoter” is a promoter capable of initiating transcription inplant cells. Exemplary plant promoters include, but are not limited to,those that are obtained from plants, plant viruses, and bacteria.Preferred promoters nay contain additional copies of one or morespecific regulatory elements, to further enhance expression and/or toalter the spatial expression and/or temporal expression of a nucleicacid molecule to which it is operably connected. For example,copper-responsive, glucocorticoid-responsive or dexamethasone-responsiveregulatory elements may be placed adjacent to a heterologous promotersequence driving expression of a nucleic acid molecule to confer copperinducible, glucocorticoid-inducible, or dexamethasone-inducibleexpression respectively, on said nucleic acid molecule. Examples ofpromoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, seeds, endosperm, embryos, fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as “tissuepreferred.” Promoters which initiate transcription only in certaintissue are referred to as “tissue specific.” A “cell type” specificpromoter primarily drives expression in certain cell types in one ormore organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue specific, tissue preferred, cell type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, anICK protein can be expressed in plant cells, bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

Means for introducing a recombinant expression vector of this inventioninto plant tissue or cells include, but are not limited to,transformation using CaCl₂ and variations thereof, in particular themethod described by Hanahan (J. Mol. Biol. 166, 557-560, 1983), directDNA uptake into protoplasts (Krens et al, Nature 296: 72-74, 1982;Paszkowski et al, EMBO J. 3:2717-2722, 1984), PEG-mediated uptake toprotoplasts (Armstrong et al, Plant Cell Reports 9: 335-339, 1990)microparticle bombardment, electroporation (Fromm et al., Proc. Natl.Acad. Sci. (USA) 82:5824-5828, 1985), microinjection of DNA (Crossway etal., Mol. Gen. Genet. 202:179-185, 1986), microparticle bombardment oftissue explants or cells (Christou et al, Plant Physiol 87: 671-674,1988; Klein et al. (1992) Biotechnology 24, 384-386),vacuum-infiltration of tissue with nucleic acid, or in the case ofplants, T-DNA-mediated transfer from Agrobacterium to the plant tissueas described essentially by An et al. (EMBO J 4:277-284, 1985),Herrera-Estrella et al. (Nature 303: 209-213, 1983a; EMBO J. 2: 987-995,1983b; In: Plant Genetic Engineering, Cambridge University Press, N.Y.,pp 63-93, 1985), or in planta method using Agrobacterium tumefacienssuch as that described by Bechtold et al., (C.R. Acad. Sci. (Paris,Sciences de la vie/Life Sciences) 316: 1194-1199, 1993) or Clough et al(Plant J. 16: 735-743, 1998). Methods for transformation ofmonocotyledonous plants are well known in the art and includeAgrobacterium-mediated transformation (Cheng et al. (1997) WO 97/48814;Hansen (1998) WO 98/54961; Hiei et al. (1994) WO 94/00977; Hiei et al.(1998) WO 98/17813; Rikiishi et al. (1999) WO 99/04618; Saito et al.(1995) WO 95/06722), microprojectile bombardment (Adams et al. (1999)U.S. Pat. No. 5,969,213; Bowen et al. (1998) U.S. Pat. No. 5,736,369;Chang et al. (1994) WO 94/1-3822; Lundquist et al. (1999) U.S. Pat. No.5,874,265/U.S. Pat. No. 5,990,390; Vasil and Vasil (1995) U.S. Pat. No.5,405,765; Walker et al. (1999) U.S. Pat. No. 5,955,362), DNA uptake(Eval et al. (1993) WO 93/181,168), microinjection of Agrobacteriumcells (von Holt 1994 DE 4309203) and sonication (Finer et at (1997) U.S.Pat. No. 5,693,512). The vector DNA may further comprise a selectablemarker gene to facilitate the identification and/or selection of cellswhich are transfected or transformed with a genetic construct. Suitableselectable marker genes contemplated herein include the ampicillinresistance (Amp^(r)), tetracycline resistance gene Tc^(r)), bacterialkanamycin resistance gene (Kan^(r)), phosphinothricin resistance gene,neomycin phosphotransferase gene (nptII), hygromycin resistance gene,β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT)gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997), andluciferase gene.

For microparticle bombardment of cells, a microparticle is propelledinto a cell to produce a transformed cell. Any suitable ballistic celltransformation methodology and apparatus can be used in performing thepresent invention. Exemplary apparatus and procedures are disclosed byStomp et al (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat.No. 4,945,050). When using ballistic transformation procedures, the geneconstruct may incorporate a plasmid capable of replicating in the cellto be transformed. Examples of microparticles suitable for use in suchsystems include 1 to 5 μm gold spheres. The DNA construct may bedeposited on the microparticle by any suitable technique, such as byprecipitation.

A whole plant may be regenerated from the transformed or transfectedcell, in accordance with procedures well known in the art. Plant tissuecapable of subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a gene construct of the presentinvention and a whole plant regenerated therefrom. The particular tissuechosen will vary depending on the clonal propagation systems availablefor, and best suited to, the particular species being transformed.Exemplary tissue targets include leaf disks, pollen, embryos,cotyledons, hypocotyls, megagametophytes, callus tissue, existingmeristematic tissue (e.g., apical meristem, axillary buds, and rootmeristems), and induced meristem tissue (e.g., cotyledon meristem andhypocotyl meristem).

The term “organogenesis”, as used herein, includes a process by whichshoots and roots are developed sequentially from meristematic centres.

The term “embryogenesis”, as used herein, includes a process by whichshoots and roots develop together in a concerted fashion (notsequentially), whether from somatic cells or gametes.

Preferably, the plant is produced according to the methods of theinvention by transfecting or transforming the plant with a geneticsequence, or by introducing to the plant a protein, by anyart-recognized means, such as microprojectile bombardment,microinjection, Agrobacterium-mediated transformation (including inplanta transformation), protoplast fusion, or electroporation, amongstothers. Most preferably the plant is produced by Agrobacterium-mediatedtransformation. Agrobacterium-mediated transformation or agrolistictransformation of plants, yeast, moulds or filamentous fungi is based onthe transfer of part of the transformation vector sequences, called theT-DNA, to the nucleus and on integration of said T-DNA in the genome ofsaid eukaryote.

The term “Agrobacterium” as used herein, includes a member of theAgrobacteriaceae, more preferably Agrobacterium or Rhizobacterium andmost preferably Agrobacterium tumefaciens.

The term “T-DNA”, or “transferred DNA”, as used herein, includes thetransformation vector flanked by T-DNA borders which is, afteractivation of the Agrobacterium vir genes, nicked at the T-DNA bordersand is transferred as a single stranded DNA to the nucleus of aneukaryotic cell.

As used herein, the terms “T-DNA borders”, “T-DNA border region”, or“border region” include either right T-DNA borders (RB) or left T-DNAborders (LB), which comprise a core sequence flanked by a border innerregion as part of the T-DNA flanking the border and/or a border outerregion as part of the vector backbone flanking the border. The coresequences comprise 22 bp in case of octopine-type vectors and 25 bp incase of nopaline-type vectors. The core sequences in the right borderregion and left border region form imperfect repeats. Border coresequences are indispensable for recognition and processing by theAgrobacterium nicking complex consisting of at least VirD1 and VirD2.Core sequences flanking a T-DNA are sufficient to promote transfer ofthe T-DNA

As used herein, the term “T-DNA transformation vector” or “T-DNA vector”includes any vector encompassing a T-DNA sequence flanked by a right andleft T-DNA border consisting of at least the right and left border coresequences, respectively, and used for transformation of any eukaryoticcell.

As used herein, the term “T-DNA vector backbone sequence” or “T-DNAvector backbone sequences” includes all DNA of a T-DNA containing vectorthat lies outside of the T-DNA borders and, more specifically, outsidethe nicking sites of the border core imperfect repeats.

The present invention includes optimized T-DNA vectors such that vectorbackbone integration in the genome of a eukaryotic cell is minimized orabsent. The term “optimized T-DNA vector” as used herein includes aT-DNA vector designed either to decrease or abolish transfer of vectorbackbone sequences to the genome of a eukaryotic cell. Such T-DNAvectors are known to the one of skill in the art and include thosedescribed by Hanson et al. (1999) Plant J. 19:727-734, by Stuiver et al.(1999—WO9901563) and by Depicker et al. (2001—WO0144482).

The current invention clearly considers the inclusion of a DNA sequenceencoding an ICK, homologue, analogue, derivative or immunologicallyactive fragment thereof as defined supra, in any T-DNA vector comprisingbinary transformation vectors, super-binary transformation vectors,co-integrate transformation vectors, Ri-derived transformation vectorsas well as in T-DNA carrying vectors used in agrolistic transformation.

As used herein, the term “binary transformation vector” includes a T-DNAtransformation vector comprising: a T-DNA region comprising at least onegene of interest and/or at least one selectable marker active in theeukaryotic cell to be transformed; and

a vector backbone region comprising at least origins of replicationactive in E. coli and Agrobacterium and markers for selection in E. coliand Agrobacterium.

The T-DNA borders of a binary transformation vector can be derived fromoctopine-type or nopaline-type Ti plasmids or from both. The T-DNA of abinary vector is only transferred to a eukaryotic cell in conjunctionwith a helper plasmid. As used herein, the term “helper plasmid”includes a plasmid that is stably maintained in Agrobacterium and is atleast carrying the set of vir genes necessary for enabling transfer ofthe T-DNA. The set of vir genes can be derived from either octopine-typeor nopaline-type Ti plasmids or from both.

As used herein, the term “super-binary transformation vector” includes abinary transformation vector additionally carrying in the vectorbackbone region a vir region of the Ti plasmid pTiBo542 of thesuper-virulent A. tumefaciens strain A281 (EP0604662, EP0687730).Super-binary transformation vectors are used in conjunction with ahelper plasmid.

As used herein, the term “co-integrate transformation vector” includes aT-DNA vector at least comprising: a T-DNA region comprising at least onegene of interest and/or at least one selectable marker active in plants;and a vector backbone region comprising at least origins of replicationactive in Escherichia coli and Agrobacterium, and markers for selectionin E. coli and Agrobacterium, and a set of vir genes necessary forenabling transfer of the T-DNA. The T-DNA borders and the set of virgenes of the T-DNA vector can be derived from either octopine-type ornopaline-type Ti plasmids or from both.

The term “Ri-derived plant transformation vector” includes a binarytransformation vector in which the T-DNA borders are derived from a Tiplasmid and the binary transformation vector being used in conjunctionwith a ‘helper’ Ri-plasmid carrying the necessary set of vir genes.

The terms “agrolistics”, “agrolistic transformation” or “agrolistictransfer” include a transformation method combining features ofAgrobacterium-mediated transformation and of biolistic DNA delivery. Assuch, a T-DNA containing target plasmid is co-delivered with DNA/RNAenabling in planta production of VirD1 and VirD2 with or without VirE2(Hansen and Chilton (1996) Proc. Natl. Acad Sci. U.S.A 93:14978-14983;Hansen et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:111726-11730;Hansen and Chilton (1997)-WO9712046).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an ICK protein.Accordingly, the invention further provides methods for producing an ICKprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding an ICK protein has beenintroduced) in a suitable medium such that an ICK protein is produced.In another embodiment, the method further comprises isolating an ICKprotein from the medium or the host cell.

The host cells of the invention can also be used to produce transgenicplant or non-human transgenic animals in which exogenous ICK sequenceshave been introduced into their genome or homologous recombinant plantsor animals in which endogenous ICK sequences have been altered. Suchplants and animals are useful for studying the function and/or activityof an ICK and for identifying and/or evaluating modulators of ICKactivity.

Trangenic Plants

As used herein, “transgenic plant” includes a plant which compriseswithin its genome a heterologous polynucleotide. Generally, theheterologous polynucleotide is stably integrated within the genome suchthat the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses as asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring event such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

A transgenic plant of the invention can be created by introducing anICK-encoding nucleic acid into the plant by placing it under the controlof regulatory elements which ensure the expression in plant cells. Theseregulatory elements may be heterologous or homologous with respect tothe nucleic acid molecule to be expressed as well with respect to theplant species to be transformed. In general, such regulatory elementscomprise a promoter active in plant cells. These promoters can be usedto modulate (e.g. increase or decrease) ICK content and/or compositionin a desired tissue. To obtain expression in all tissues of a transgenicplant, preferably constitutive promoters are used, such as the 35 Spromoter of CaMV (Odell, Nature 313 (1985), 810-812) or promoters fromsuch genes as rice actin (McElroy et al. (1990) Plant Cell 2:163-171)maize H3 histone (Lepetit et al. (1992) Mol. Gen. Genet 231:276-285) orpromoters of the polyubiquitin genes of maize (Christensen, Plant Mol.Biol. 18 (1982), 675-689). In order to achieve expression in specifictissues of a transgenic plant it is possible to use tissue specificpromoters (see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251 or Table 5,below).

TABLE 5 EXPRESSION GENE SOURCE PATTERN REFERENCE I: CELL-SPECIFIC,TISSUE-SPECIFIC, AND ORGAN-SPECIFIC PROMOTERS α-amylase aleurone Lanahanet al, Plant Cell 4:203- (Amy32b) 211, 1992; Skriver et al, Proc NatlAcad Sci USA 88:7266- 7270, 1991 cathepsin β-like aleurone Cejudo et al,Plant Mol Biol gene 20:849-856, 1992 Agrobacterium camblum Nilsson etal, Physiol Plant rhizogenes rolB 100:456-462, 1997 AtPRP4 flowershttp://salus.medium.edu/mmg/ tierney/html chalcone synthase flowers Vander Meer et al, Plant Mol (chsA) Biol 15:95-109, 1990 LAT52 anther Twellet al, Mol Gen Genet 217:240-245, 1989 apetala-3 flowers chitinase fruit(berries, Thomas et al. CSIRO Plant grapes, etc) Industry, Urrbrae,South Australia, Australia; http://winetitles.com.au/gwrdc/ csh95-1.htmlrbcs-3A green tissue (eg Lam et al, Plant Cell 2:857-866, leaf) 1990;Tucker et al., Plant Physiol 113:1303-1308, 1992 leaf-specific leafBaszczynski et al, Nucl Acid genes Res 16:4732, 1988 AtPRP4 leafhttp://salus.medium.edu/mmg/ tierney/html chlorella virus leaf Mitra andHiggins, Plant Mol adenine methyl- Biol 26:85-93, 1994 transferase genepromoter aldP gene leaf Kagaya et al, Mol Gen Genet promoter from248:668-674, 1995 rice rbcs promoter leaf Kyozuka et al, Plant Physiolfrom rice or 102:991-1000, 1993 tomato Pinus cab-6 leaf Yamamoto et al,Plant Cell Physiol 35:773-778, 1994 rubisco promoter leaf cab(chlorophyll leaf a/b/binding protein pea Blec4 gene vegetative andfloral Mandaci and Dobres, Plant Mol epidermal tissues Biol 34:961-965SAM22 senescent leaf Crowell et al, Plant Mol Biol 18:459-466, 1992 ltpgene (lipid Fleming et al, Plant J 2:855-862, transfer gene) 1992 R.japonicum nif nodule U.S. Pat. No. 4,803,165 gene B. japonicum noduleU.S. Pat. No. 5,008,194 nifH gene GmENOD40 nodule Yang et al, Plant J3:573-585, 1993 PEP carboxylase nodule Pathirana et al, Plant Mol Biol(PEPC) 20:437-450, 1992 leghaemoglobin nodule Gordon et al, J Exp Bot(Lb) 44:1453-1465, 1993 Tungro phloem Bhattacharyya-Pakrasi et al,bacilliform virus Plant J 4:71-79, 1992 gene pollen-specific pollen;microspore Albani et al, Plant Mol Biol genes 15:605, 1990; Albani etal, Plant Mol Biol 16:501, 1991 Zm13 pollen Guerrero et al, Mol GenGenet 224:161-168, 1993 apg gene microspore Twell et al, Sex PlantReprod 6:217-224, 1993 maize pollen- pollen Hamilton et al, Plant MolBiol specific gene 18:211-218, 1992 sunflower pollen- pollen Baltz etal, Plant J 2:713-721, expressed gene 1992 B. napus pollen- pollen;anther; Amoldo et al, J Cell Biochem, specific gene tapetum Abstract No.Y101, 204, 1992 root-expressible roots Tingey et al, EMBO J 6:1, 1987genes tobacco auxin- root tip Van der Zaal et al, Plant Mol induciblegene Biol 16:983, 1991 β-tubulin root Oppenheimer et al, Gene 63:87,1988 tobacco root- root Conkling et al, Plant Physiol specific genes93:1203, 1990 B. napus G1-3b root U.S. Pat. No. 5,401,836 gene SbPRP1roots Suzuki et al, Plant Mol Biol 21:109-119, 1993 AtPRP1; AtPRP3roots; root hairs http://salus.medium.edu/mmg/ tierney/html RD2 generoot cortex http://2.cnsu.edu/ncsu/research TobRB7 gene root vasculaturehttp://2.cnsu.edu/ncsu/research AtPRP4 leaves; flowers;http://salus.medium.edu/mmg/ lateral root tierney/html primordiaseed-specific seed Simon et al, Plant Mol Biol genes 5:191, 1985;Scofield et al, J Biol Chem 262:12202, 1987; Baszczynski et al, PlantMol Biol 14:633, 1990 Brazil Nut seed Pearson et al, Plant Mol Biolalbumin 18:235-245, 1992 legumin seed Ellis et al, Plant Mol Biol10:203-214, 1988 glutelin (rice) seed Takaiwa et al, Mol Gen Genet208:15-22, 1986; Takaiwa et al, FEBS Lett 221:43-47, 1987 zein seedMatzke et al, Plant Mol Biol 14:323-32 1990 napA seed Stalberg et al,Planta 199:515- 519, 1996 wheat LMW and endosperm Mol Gen Genet216:81-90, HMW glutenin-1 1989; Nucl Acids Res 17:461-462, 1989 wheatSPA seed Albanl et al, Plant Cell 9:171- 184, 1997 cZ19B1, maize seedWO0011177 19 kDa zein ml1ps, maize seed WO0011177 myoinositol-1-Pisynthase wheat α, β, endosperm EMBO J 3:1409-1415, 1984 γ-gliadinsbarley ltr1 endosperm promoter barley B1, C, D, endosperm Theor Appl Gen98:1253-1262, hordein 1999; Plant J 4:343-355, 1993; Mol Gen Genet250:750-60, 1996 barley DOF endosperm Mena et al, Plant J 116:53-62,1998 blz2 endosperm EP99106056.7 synthetic endosperm Vicente-Carbajosaet al, Plant J promoter 13:629-640, 1998 rice prolamin endosperm Wu etal, Plant Cell Physiol NRP33 39:885-889, 1998 rice α-globulin endospermWu et al, Plant Cell Physiol Glb-1 39:885-889, 1998 maize END genesendosperm WO0012733 barley END1 endosperm WO9808961 barley NUC1 nucellusWO9808961 rice OSH1 embryo Sato et al, Proc Natl Acad Sci USA93:8117-8122, 1996 rice α-globulin endosperm Nakase et al, Plant MolBiol REB/OHP-1 33:513-522, 1997 rice ADP-glucose endosperm Trans Res6:157-168, 1997 PP maize ESR gene endosperm Plant J 12:235-246, 1997family sorgum γ-kafirin endosperm Plant Mol Biol 32:1029-1035, 1996 KNOXembryo Postma-Haarsma et al, Plant Mol Biol 39:257-271, 1999 riceoleosin embryo and aleuron Wu et al, J Biochem 123:386, 1998 sunfloweroleosin seed (embryo and Cummins et al, Plant Mol Biol dry seed)19:873-876, 1992 LEAFY shoot meristem Weigel et al, Cell 69:843-859,1992 Arabidopsis shoot meristem Accession number AJ131822 thaliana knat1Malus domestica shoot meristem Accession number Z71981 kn1 CLAVATA1shoot meristem Accession number AF049870 stigma-specific stigmaNasrallah et al, Proc Natl Acad genes Sci USA 85:5551, 1988; Trick etal, Plant Mol Biol 15:203, 1990 class I patatin tuber Liu et al, PlantMol Biol gene 153:386-395, 1991 PCNA rice meristem Kosugi et al, NuclAcids Res 19:1571-1576, 1991; Kosugi and Ohashi, Plant Cell 9:1607-1619,1997 Pea TubA1 Dividing cells Stotz and Long, Plant Mol Biol tubulin41:601-614, 1999 Arabidopsis cycling cells Chung and Parish, FEBS Lettcdc2a 362:215-219, 1995 Arabidopsis Anthers; mature Li et al, PlantPhysiol Rop1A pollen + pollen 118:407-417, 1998 tubes ArabidopsisMeiosis-associated Klimyuk and Jones, Plant J AtDMC1 11:1-14, 1997 PeaPS-IAA4/5 Auxin-inducible Wong et al, Plant J 9:587-599, and PS-IAA61996 Pea farnesyltrans- Meristematic Zhou et al, Plant J 12:921-930,ferase tissues; phloem near 1997 growing tissues; light- and sugar-repressed Tobacco (N. Dividing cells/ Trehin et al, Plant Mol.Biol.sylvestris) cyclin meristematic tissue 35:667-672, 1997 B1;1Catharanthus Dividing cells/ Ito et al, Plant J 11:983-992, roseusMitotic meristematic tissue 1997 cyclins CYS (A-type) and CYM (B-type)Arabidopsis Dividing cells/ Shaul et al, Proc Natl Acad Sci cyc1At (=cycmeristematic tissue USA 93:4868-4872, 1996 B1;1) and cyc3eAt (A-type)Arabidopsis tef1 Dividing cells/ Regad et al, Mol Gen Genet promoter boxmeristematic tissue 248:703-711, 1995 Catharanthus Dividing cells/ Itoet al, Plant Mol Biol roseus cyc07 meristematic tissue 24:863-878, 1994II: CONSTITUTIVE PROMOTERS Actin constitutive McElroy et al, Plant Cell2:163-171, 1990 CAMV 35S constitutive Odell et al, Nature 313:810-812,1985 CaMV 19S constitutive Nilsson et al, Physiol Plant 100:456-462,1997 GOS2 constitutive de Pater et al, Plant J 2:837-844, 1992 ubiquitinconstitutive Christensen et al, Plant Mol Biol 18:675-689, 1992 ricecyclophilin constitutive Buchholz et al, Plant Mol Biol 26:837-843, 1994maize histone H3 constitutive Lepetit et al, Mol Gen Genet 231:276-285,1992 alfalfa histone H3 constitutive Wu et al, Nucleic Acids Res17:3057-3063, 1989; Wu et al, Plant Mol Biol 11:641-649, 1988 actin 2constitutive An et al, Plant J 10:107-121, 1996 III: STRESS-INDUCIBLEPROMOTERS P5CS (delta(1)- salt, water Zhang et al, Plant Scipyrroline-5-car- 129:81-89, 1997 boxylate syntase) cor15a cold Hajela etal, Plant Physiol 93:1246-1252, 1990 cor15b cold Wlihelm et al, PlantMol Biol 23:1073-1077, 1993 cor15a (−305 cold, drought Baker et al,Plant Mol Biol to +78 nt) 24:01-713, 1994 rd29 salt, drought, coldKasuga et al, Nature Biotechnol 18:287-291, 1999 heat shock heat Barroset al, Plant Mol Biol 19 proteins, includ- 665-75, 1992. Marrs et al,Dev ing artificial pro- Genet14:27-41, 1993. Schoffi et moterscontaining al, Mol Gen Genet 217:246-53, the heat shock 1989. element(HSE) smHSP (small heat Waters et al, J Exp Bot heat shock 47:325-338,1996 proteins) wcs120 cold Ouellete et al, FEBS Lett 423:324-328, 1998ci7 cold Kirch et al, Plant Mol Biol 33:897-909, 1997 Adh cold, drought,Dolferus et al, Plant Physiol hypoxia 105:1075-87, 1994 pwsi18 water:salt and Joshee et al, Plant Cell Physiol drought 39:64-72, 1998 ci21Acold Schneider et al, Plant Physiol 113:335-45, 1997 Trg-31 droughtChaudhary et al, Plant Mol Biol 30:1247-57, 1996 Osmotin osmoticRaghothama et al, Plant Mol Biol 23:1117-28, 1993 LapA wounding,WO99/03977 University of enviromental California/INRA IV:PATHOGEN-INDUCIBLE PROMOTERS RB7 Root-knot U.S. Pat. No. 5,760,386 -North nematodes Carolina State University; (Meloidogyne spp.) Oppermanet al, Science 263:221-23, 1994 PR-1, 2, 3, 4, 5, fungal, viral, Ward etal, Plant Cell 8, 11 bacterial 3:1085-1094, 1991; Reiss et al 1996;Lebel et al, Plant J 16:223-233, 1998; Melchers et al, Plant J5:469-480, 1994; Lawton et al, Plant Mol Biol, 19:735-743, 1992 HMG2nematodes WO9503690 - Virginia Tech Intellectual Properties Inc. Abi3Cyst nematodes unpublished (Heterodera spp.) ARM1 nematodes Barthels etal, Plant Cell 9:2119-2134, 1997 WO 98/31822 - Plant Genetic SystemsAtt0728 nematodes Barthels et al, Plant Cell 9:2119-2134, 1997PCT/EP98/07761 Att1712 nematodes Barthels et al, Plant Cell 9,2119-2134,1997 PCT/EP98/07761 Gst1 Different types of Strittmatter et al, MolPlant- pathogens Microbe Interact 9:68-73, 1996 LEMMI nematodes WO92/21757 - Plant Genetic Systems CLE geminivirus PCT/EP99/03445 -CINESTAV PDF1.2 Fungal including Manners et al, Plant Mol Biol,Alternaria 38:1071-1080, 1998 brassicicola and Botrytis cinerea Thi2.1Fungal-Fusarium Vignutelli et al, Plant J oxysporum f sp. 14:285-295,1998 matthiolae DB#226 nematodes Bird and Wilson, Mol Plant- MicrobeInteract 7:419-442, 1994 WO 95.322888 DB#280 nematodes Bird and Wilson,Mol Plant- Microbe Interact 7:419-442, 1994 WO 95.322888 Cat2 nematodesNiebel et al, Mol Plant-Microbe Interact 8:371-378, 1995 □Tub nematodesAristizabal et al (1996), 8^(th) International Congress on Plant-Microbe Interaction, Knoxville US B-29 sHSP nematodes Fenoll et al(1997) in: Cellular and molecular aspects of plant- nematodeinteractions. Kluwer Academic, C. Fenoll, F. M. W. Grundler and S. A.Ohl (Eds.), Tsw12 nematodes Fenoll et al (1997) in: Cellular andmolecular aspects of plant- nematode interactions. Kluwer Academic, C.Fenoll, F. M. W. Grundler and S. A. Ohl (Eds.), Hs1(pro1) nematodes WO98/122335 - Jung nsLTP viral, fungal, Molina and Garcia-Olmedo bacterialFEBS Lett, 316:119-122, 1993 RIP viral, fungal Turner et al, Proc NatlAcad Sci USA 94:3866-3871, 1997

The promoters listed in the foregoing table are provided for thepurposes of exemplification only and the present invention is not to belimited by the list provided therein. Those skilled in the art willreadily be in a position to provide additional promoters that are usefulin performing the present invention. The promoters listed may also bemodified to provide specificity of expression as required.

Known are also promoters which are specifically active in tubers ofpotatoes or in seeds of different plants species, such as maize, Vicia,wheat, barley and the like. Inducible promoters may be used in order tobe able to exactly control expression under certain environmental ordevelopmental conditions such as pathogens, anaerobia, or light.Examples of inducible promoters include the promoters of genes encodingheat shock proteins or microspore-specific regulatory elements(WO96/16182). Furthermore, the chemically inducible Tet-system may beemployed (Gatz, Mol. Gen. Genet 227 (1991); 229-237). Further suitablepromoters are known to the person skilled in the art and are described,e.g., in Ward (Plant Mol. Biol. 22 (1993), 361-366). The regulatoryelements may further comprise transcriptional and/or translationalenhancers functional in plants cells. Furthermore, the regulatoryelements may include transcription termination signals, such as a poly-Asignal, which lead to the addition of a poly A tail to the transcriptwhich may improve its stability.

In the case that a nucleic acid molecule according to the invention isexpressed in the sense orientation, the coding sequence can be modifiedsuch that the protein is located in any desired compartment of the plantcell, e.g., the nucleus, endoplasmatic reticulum, the vacuole, themitochondria, the plastids, the apoplast, or the cytoplasm.

Methods for the introduction of foreign DNA into plants are also wellknown in the art. These include, for example, the transformation ofplant cells or tissues with T-DNA using Agrobacterium tumefaciens orAgrobacterium rhizogenes, the fusion of protoplasts, direct genetransfer (see, e.g., EP-A 164 575), injection, electroporation,biolistic methods like particle bombardment, pollen-mediatedtransformation, plant RNA virus-mediated transformation,liposome-mediated transformation, transformation using wounded orenzyme-degraded immature embryos, or wounded or enzyme-degradedembryogenic callus and other methods known in the art. The vectors usedin the method of the invention may contain further functional elements,for example “left border”- and “right border”-sequences of the T-DNA ofAgrobacterium which allow for stably integration into the plant genome.Furthermore, methods and vectors are known to the person skilled in theart which permit the generation of marker free transgenic plants, i.e.,the selectable or scorable marker gene is lost at a certain stage ofplant development or plant breeding. This can be achieved by, forexample, cotransformation (Lyznik, Plant Mol. Biol. 13 (1989), 151-161;Peng, Plant Mol. Biol. 27 (1995), 91-104) and/or by using systems whichutilize enzymes capable of promoting homologous recombination in plants(see, e.g., WO97/08331; Bayley, Plant Mol. Biol. 18 (1992), 353-361);Lloyd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet.230 (1991), 170-176; Onouchi, Nucl. Acids Res. 19 (1991), 6373-6378).Methods for the preparation of appropriate vectors are described by,e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition(1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Suitable strains of Agrobacterium tumefaciens and vectors, as well astransformation of Agrobacteria, and appropriate growth and selectionmedia are described in, for example, GV3101 (pMK90RK), Koncz, Mol. Gen.Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. AcidRes. 13 (1985), 4777; Bevan, Nucleic. Acid Res. 12(1984), 8711; Koncz,Proc. Natl. Acad. Sci. USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol.20 (1992), 963-976; Koncz, Specialized vectors for gene tagging andexpression studies. In: Plant Molecular Biology Manual Vol 2, Gelvin andSchilperoort (Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ.(1994), 1-22; EP-A-120 516; Hoekema: The Binary Plant Vector System,Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V, Fraley,Crit. Rev. Plant. Sci., 4, 1-46; An, EMBO J. 4 (1985), 277-287).Although the use of Agrobacterium tumefaciens is preferred in the methodof the invention, other Agrobacterium strains, such as Agrobacteriumrhizogenes, may be used, for example, if a phenotype conferred by saidstrain is desired.

Methods for the transformation using biolistic methods are known to theperson skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994),37-48; Vasil, Bio/Technology 11 (1993), 1553-1558 and Christou (1996)Trends in Plant Science 1, 423-431. Microinjection can be performed asdescribed in Potrykus and Spangenberg (eds.), Gene Transfer To Plants.Springer Verlag, Berlin, N.Y. (1995).

The transformation of most dicotyledonous plants may be performed usingthe methods described above or using transformation via biolisticmethods as, e.g. described above as well as protoplast transformation,electroporation of partially permeabilized cells, or introduction of DNAusing glass fibers.

In general, the plants which are modified according to the invention maybe derived from any desired plant species. They can be monocotyledonousplants or dicotyledonous plants, preferably they belong to plant speciesof interest in agriculture, wood culture or horticulture interest, suchas crop plants (e.g., maize, rice, barley, wheat, rye, oats), potatoes,oil producing plants (e.g., oilseed rape, sunflower, pea nut, soy bean),cotton, sugar beet, sugar cane, leguminous plants (e.g., beans, peas),or wood producing plants, preferably trees.

The present invention also relates to a transgenic plant cell whichcontains (preferably stably integrated into its genome) a nucleic acidmolecule of the present invention linked to regulatory elements whichallow expression of the nucleic acid molecule in plant cells. Thepresence and expression of the nucleic acid molecule in the transgenicplant cells leads to the synthesis of an ICK protein and may lead tophysiological and phenotypic changes in plants containing such cells.

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerwhich has been introduced with a polynucleotide of the presentinvention.

Plant cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmiliilan Publishing Company, New York, pp. 124-176 (1983); andBinding, Regeneration of Plants, Plant Protoplasts, CRC Press, BocaRaton, pp. 21-73 (1985).

Transformed plant cells, calli or explant can be cultured onregeneration medium in the dark for several weeks, generally about 1 to3 weeks to allow the somatic embryos to mature. Preferred regenerationmedia include media containing MS salts, such as PHI-E and PHI-F media.The plant cells, calli or explant are then typically cultured on rootingmedium in a light/dark cycle until shoots and roots develop. Methods forplant regeneration are known in the art and preferred methods areprovided by Kamo et al, (Bot. Gaz. 146(3):324-334, 1985), West et al,(The Plant Cell 5:1361-1369. 1993), and Duncan et al. (Planta165:322-332, 1985).

Small plantlets can then be transferred to tubes containing rootingmedium and allowed to grow and develop more roots for approximatelyanother week. The plants can then be transplanted to soil mixture inpots in the greenhouse.

The regeneration of plants containing the foreign gene introduced byAgrobacterium from leaft explants can be achieved as described by Horschet al., Science, 227:1229-1231 (1985). In this procedure, transformantsare grown in the presence of a selection agent and in a medium thatinduces the regeneration of shoots in the plant species beingtransformed as described by Fraley et al., Proc. Natl. Acad. Sci, USA.80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Klee et al., Ann. Rev. of Plant Phys., 38:467-486 (1987). Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, from example, Methods for PlantMolecular Biology, A. Weissbach and H. Weissback, eds., Academic Press,Inc., San Diego, Calif. (1988). This regeneration and growth processincludes the steps of selection of transformant cells and shoots,rooting ht transformant shoots and growth of the plantlets in soil. Formaize cell culture and regeneration see generally, The Maize Handbook,Freeling and Walbot, Eds., Springer, New York (1994); Corn and CornImprovement, 3rd edition, Sprague and Dudley Eds., American Society ofAgronomy, Madison, Wis. (1988).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype, (e.g., altered cell cycle content or composition).

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences.

Transgenic plants expressing the selectable marker can be screened fortransmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Transgeniclines are also typically evaluated on levels of expression of theheterologous nucleic acid. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then analyzed for protein expression by Western immunoblot analysisusing the specifically reactive antibodies of the present invention. Inaddition, in situ hybridization and immunocytochemistry according tostandard protocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

A preferred embodiment of the invention is a transgenic plant that ishomozygous for the added heterologous nucleic acid; i.e., a transgenicplant that contains two added nucleic acid sequences, one gene at thesame locus on each chromosome of a chromosome pair. A homozygoustransgenic plant can be obtained by sexually mating (selfing) aheterozygous transgenic plant that contains a single added heterologousnucleic acid, germinating some of the seed produced and analyzing theresulting plants produced for altered cell division relative to acontrol plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

The present invention also relates to transgenic plants and plant tissuecomprising transgenic plant cells according to the invention. Due to the(over)expression of an ICK molecule, e.g., at developmental stagesand/or in plant tissue in which they do not naturally occur, thesetransgenic plants may show various physiological, developmental and/ormorphological modifications in comparison to wild-type plants.

Therefore, part of this invention is the use of the ICK molecules tomodulate the cell cycle and/or plant cell division and/or growth inplant cells, plant tissues, plant organs and/or whole plants. To thescope of the invention also belongs a method for influencing theactivity of cell cycle control proteins such as CDKs and cyclins in aplant cell by transforming the plant cell with a nucleic acid moleculeaccording to the invention and/or manipulation of the expression of themolecule.

Furthermore, the invention also relates to a transgenic plant cell whichcontains (preferably stably integrated into its genome) a nucleic acidmolecule of the invention or part thereof, wherein the transcriptionand/or expression of the nucleic acid molecule or part thereof leads toreduction of the synthesis of an ICK. In a preferred embodiment, thereduction is achieved by an anti-sense, sense, ribozyme, co-suppressionand/or dominant mutant effect. The reduction of the synthesis of aprotein according to the invention in the transgenic plant cells canresult in an alteration in, e.g., cell division. In transgenic plantscomprising such cells this can lead to various physiological,developmental and/or morphological changes.

In yet another aspect, the invention relates to harvestable parts and topropagation material of the transgenic plants of the invention whicheither contain transgenic plant cells expressing a nucleic acid moleculeaccording to the invention or which contain cells which show a reducedlevel of the described protein. Harvestable parts can be in principleany useful parts of a plant, for example, flowers, pollen, seedlings,tubers, leaves, stems, fruit, seeds, roots etc. Propagation materialincludes, for example, seeds, fruits, cuttings, seedlings, tubers,rootstocks, and the like.

VI. Agricultural Compositions

The ICK nucleic acid molecules, ICK proteins, and anti-ICK antibodies(also referred to herein as “active compounds”) of the invention can beincorporated into compositions useful in agriculture and in plant celland tissue culture. Plant protection compositions can be prepared byconventional means commonly used for the application of, for example,herbicides and pesticides. For example, certain additives known to thoseskilled in the art stabilizers or substances which facilitate the uptakeby the plant cell, plant tissue or plant may be used.

The agricultural compositions can be included in a container, pack, ordispenser together with instructions for administration.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing areincorporated herein by reference.

EXAMPLES

Unless stated otherwise in the example, all recombinant DNA techniquesare performed according to protocols as described in Sambrook et al.(1989), Molecular cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press, NY or in Ausubel et al. (1999) Current Protocols inMolecular Biology, CD-ROM, John Wiley & Sons, Inc, NY. Standard materialand methods for plant molecular work are described in Plant MolecularBiology Labfase (1993) by R. D. D. Croy, jointly published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications(UK).

Example 1 Construction of the Rice Two-Hybrid cDNA Library

For the identification of ICKs, a two-hybrid system based on Gal4transcriptional activation was developed with the aim of identifyingCDC2Os1-interacting proteins. Total RNA was extracted from cellsuspension cultures harvested at 0, 3, 6, 9, and 12 days aftersubculturing in fresh medium. These timepoints correspond to cells inthe lag phase following subculture, the exponential growth, and the latestationary phase. Equimolar amounts of total RNA (5×100 μg) from thedifferent fractions were then pooled. The total RNA sample was used topurify 5 μg of polyA+mRNA using the Poly(A) Quik mRNA Isolation kit(Stratagene), based on oligo(dT) cellulose columns. Synthesis andsubcloning of the cDNA into the HybriZAP-2.1 lambda vector wereperformed according to the manufacturer's guidelines (Stratagene).Approximately 2 million independent plaque-forming units were produced,with an average insert size of 1.0 kb. Bank amplification and massexcision to obtain phagemids used to transform yeast were done followingthe same instruction manual.

The rice Cdc2-Os1 gene was amplified using the following primers (sense:5′-AGGGATGTTTAATACCACTAC-3′, SEQ ID NO:33 and antisense primer:5′-GCACAGTTGAAGTGAACTTGC-3′, SEQ ID NO:34) and the Pfx DNA polymerase(Promega). The PCR fragment was blunt end cloned into the SmaI site ofpBD-Gal4 (Stratagene) bait vector in frame with the binding domain. Thebait vector was introduced in yeast PJ69-4a according to the “Quick andEasy TRAFO Protocol” (Gietz, R. D. and R. A. Woods, (1994) HighEfficiency transformation in Yeast (Invited Book Chapter) In: MolecularGenetics of Yeast: Practical Approaches, ed. J. A. Johnston, OxfordUniversity Press pp. 121-134) and the transformants selected on dropoutmedium lacking tryptophane. These transformants were confirmed by PCRusing the above mentioned primers.

Using the yeast two-hybrid assay, a number of Cdc2Os1-interacting cloneswere identified. Surprisingly, however, none of these clones was an ICK.

The lambdaHybriZAP-2.1 (Stratagene, La Jolla, Calif.) two-hybrid cDNAlibrary made from the rice suspension culture as outlined above wasdeposited on Oct. 27, 2000 with the ‘Belgian co-ordinated collections ofmicro-organisms’ (BCCM-LMBP, University Gent, Laboratorium voorMoleculaire Biologie, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium)according to the regulations of the Budapest Treaty. BCCM is recognizedby the WIPO as an IDA. The accession number of the deposit is LMBP4268).

Example 2 Molecular Cloning of ICK Fragments

To overcome the aforementioned problem, public sequence databases(available at, for example, http://www.ncbi.nlm.nih.gov,http://www.tigr.org) were screened for the “GRYEW” amino acid motiflocated at the carboxy-termini of all known plant ICKs, using theadvanced BLAST program with a high expect value (1000).

Using the foregoing database mining approach, four hits were obtained inOryza sativa, one EST (partial cDNA, GenBank accession number AU075786,SEQ ID NO:1, termed OsICK2), two GSS (partial genomic sequences, GenBankaccession number AQ574895, SEQ ID NO:2, termed OsICK1 and GenBankaccession number AQ365042, SEQ ID NO:3, termed OsICK3), and one HTGS(high throughput genomic sequences, GenBank accession number AC069145,SEQ ID NO:4, termed OsICK4), none of them annotated as encoding aputative ICK protein. Four additional unannotated clones, two Zea maysESTs (GenBank accession number AI737717, SEQ ID NO:5, termed ZmICK1; andGenBank accession number AW267370, SEQ ID NO:6, termed ZmICK2) a Sorghumbicolor BAC genomic clone (GenBank accession number AF061282, SEQ IDNO:7, termed SbICK), and a Pinus taeda cDNA (GenBank accession numberAA556411, SEQ ID NO:8, termed PtICK) were identified as plant ICKs.

The following Primers were designed to perform a PCR to amplify theidentified fragments:

OsICK1 yielding a 178 bp DNA fragment:5′-TAACTCGATCCCCAGCCTCTCCCA-3′ and (SEQ ID NO: 25)5′-TACAATTACGACATTGCCCTCGAC-3′ (SEQ ID NO: 26) OsICK2 yielding a 430 bpfragment: 5′-CCGCCGAGATCGAGGCGTTCTTCG-3′ and (SEQ ID NO: 27)5′-AAACCTCTGATAAATACTGGGACG-3′ (SEQ ID NO: 28) OsICK3 yielding a 200 bpfragment: 5′-CTGTCACACACTCACACTCACACT-3′ and (SEQ ID NO: 29)5′-CGAAGAACGCCTCGATCTCC-3′ (SEQ ID NO: 30) OsICK4 yielding a 271 bpfragment: 5′-GAATACCAGGGAGACGACACCTTGC-3′ (SEQ ID NO: 31)5′-TCAGTCTAGGTTGACCCATTCAAAC-3′ (SEQ ID NO: 32)

The two-hybrid bank (in the form of plasmid) was used to amplify all ofthese fragments, which were then subcloned into the pUC18 SmaI site andtransformed info E. coli following standard molecular biologytechniques.

Example 3 Hybridization Screening of the Rice Two-Hybrid cDNA Libraryand Molecular Cloning of Full-Length OsICK2 cDNA

Following plasmid purification from E. coli, the four fragments wereisolated by restriction and gel-purified following art known techniques.The respective fragments were radioactively labelled with the aim toidentify, via homologous hybridisation, the corresponding cDNAs in thetwo-hybrid library. Serial dilutions of a plasmid preparation (6 μg/μl)of the library was used as template for PCR amplification. For OsICK2,the 430 bp could be amplified until dilution 10-4. This dilution wasused to transform XL-10 Gold (Stratagene) ultracompetent cells.Approximately 70,000 colonies were then screened with the labelledOsICK2 fragment. A number of putative positives colonies werere-screened via PCR using the OsICK2 primers. A total of 5 positiveclones were finally obtained, one of which was further characterized.Sequencing of the insert indicated that this clone was indeed the OsICK2fall length clone.

Example 4 Two Hybrid Screening and Hybridization Screening of the RiceTwo-Hybrid cDNA Library and Molecular Cloning of Full-Length OsICK4 cDNA

As described in Example 1, a first two-hybrid screening of the ricetwo-hybrid cDNA library using Cdc2-Os1 as bait did not yield any OsICKsas Cdc2-Os1 interactors. The two-hybrid screening was repeated in asecond attempt to obtain OsICK clones via this methodology. In parallel,hybridization screening of the rice two-hybrid cDNA library wasperformed as described in Example 3 with the aim of obtaining afull-length OsICK4 cDNA.

Two-Hybrid Screening

Screening of a library prepared from an actively dividing rice cellsuspension was accomplished by sequential transformation of yeast.PJ69-4a carrying pBD-cdc2Os-Gal4 was transformed with library DNAaccording to the protocol Matchmaker Two-Hybrid System(protocol#PT1020-1, version#PR4Y411) from CLONTECH Laboratories, Inc.After a library scale transformation (efficiency 17268 clones/μg DNA) atotal number of 672 clones were recovered from selective medium (SD-TLH)lacking tryptophane, leucine and histidine. These clones were furtherselected on SD-TLA (lacking tryptophane, leucine and adenine) andSD-TLAH (lacking tryptophane, leucine, adenine and histidine). Nineclones were finally selected and sequenced. Among the nine clones a CDKinhibitor was identified which was a putative partial OsICK4 cDNAsequence missing the 5′ end.

Hybridization Screening

The OsICK4 fragment cloned in pUC 18 as described in Example 2 was usedto prepare a probe for the hybridization screening of the ricetwo-hybrid cDNA library. The primers with SEQ ID NO:31 and SEQ ID NO:32(see Example 2) were used for this purpose. Approximately 750.000plaque-forming units from the cell suspension two-hybrid cDNA librarywere screened. Plaques were transferred to Hybond N+ membranes(Amersham). The filters obtained were prehybridized in sodium phosphate0.25M, (pH 7.2), SDS 7% at 60° C. for 4 hours. Hybridization wasperformed with the prehybridization buffer containing 50 ng of[α]³²P-dCTP-labelled probe at 60° C. overnight (protocol of Church G. M.and Gilbert W. PNAS USA 81:1991-1994). The filters were washed twicewith 1×SSC, 0.1% SDS at 60° C. for 30 minutes and then once with0.1×SSC, 0.1% SDS at 60° C. for 30 minutes. The membranes were placedinto a film cassette and exposed to film for 6 hours. Four clones wereisolated. A second round of screening on these clones was performed.Pure positive plaques were isolated from which the phagemids wereexcised. Two clones were obtained for sequencing. One of them is a filllength clone with a size of 1.1 kb. This clone contains an ORF of 585 bpencoding a protein of 194 amino acids. This ORF comprises the partialOsICK4 nucleotide sequence as identified in the two-hybrid screening(supra; see FIG. 10).

The nucleotide sequence of this full-length OsICK4 is depicted in FIG.1B and set forth as SEQ ID NO:43. The amino acid sequence of thisfull-length OsICK4 is depicted in FIG. 11 and set forth as SEQ ID NO:44.The OsICK4 genomic sequence was also determined as is set forth hereinin SEQ ID NO:45 and depicted in FIG. 18.

On Sep. 30, 2000, the fifth version of GenBank Accession Number AC069145was released in GenBank. GenBank Accession Number AC069145 waspreviously identified by the inventors as containing a putative OsICKsequence (see Example 2). Contrary to previous versions, AC069145.5 nowhas the putative OsICK annotated as a putative cyclin-dependent kinaseinhibitor identified by protein ID=AAG16867.1. The ORF prediction and,thus, the predicted protein sequence is, however, different from thecDNA and protein sequences experimentally determined by the presentapplication. The OsICK protein predicted in GenBank (protein ID=AAG16867.1) contains an insertion of 48 superfluous amino acids and is,therefore, almost certainly functionally inactive. The comparison of theGenBank predicted protein and the currently experimentally obtainedprotein is set forth herein in FIG. 11.

The current experiments also clearly demonstrate the physicalinteraction of the rice cyclin-dependent kinase Cdc2-Os1 with the ricecyclin-dependent kinase inhibitor OsICK4 as a partial clone of thelatter was obtained via two-hybrid screening of a rice cDNA library.These data furthermore indicate that the NH₂-terminal 66 amino acids ofthe OsICK4 are apparently not required for interaction with Cdc2-Os1.

Example 5 Characterization of OsICK2 and OsICK4

As described in the preceding example, screening of the rice two-hybridcDNA library resulted in the identification of the full length OsICK2cDNA (SEQ ID NO:9) encoding a full-length rice OsICK2 protein (SEQ IDNO:10) and of the full length OsICK4 cDNA (SEQ ID NO:43) encoding afull-length rice OsICK4 protein (SEQ ID NO:44). The partial open readingframe of SEQ ID NO:1 encodes, when taking into account a frame shift atnucleotide 52 of SEQ ID NO:1, the carboxy-terminal part of the OsICK2protein. When comparing the nucleotide sequences SEQ ID NO:1 and SEQ IDNO:9, however, a stretch of 104 nucleotides (nucleotides 1121-1224 inSEQ ID NO:9) can be discerned in the 3′ untranslated region of SEQ IDNO:9 which are not present in SEQ ID NO:1 (see FIG. 1). This indicatesthat OsICK2 primary transcripts are prone to alternative polyadenylationsite selection. A closer examination of the 3′ untranslated region inSEQ ID NO:9 reveals the presence of two canonical mRNA polyadenylationsites (Edwards-Gilbert et al. 1997), namely AAUAAA (nucleotides 922-927in SEQ ID NO:9) and AUUAAA (nucleotides 1156-1161 in SEQ ID NO:9). Thesecond of these sites is not present in the 3′ untranslated region ofSEQ ID NO:1 which is consistent with the use of alternativepolyadenylation sites in primary OsICK2 transcripts.

Another interesting feature of the 3′ untranslated regions of the twodifferent transcripts most likely derived from the same OsICK2 geneconcerns class I AREs (AU-rich elements). Transcripts corresponding toSEQ ID NO:1 comprise one such ARE (AUUUA, nucleotides 419-423 in SEQ IDNO:1) whereas transcripts corresponding to SEQ ID NO:9 contain two AREs(AUUUA; nucleotides 1076-1080 and 1161-1165 in SEQ ID NO:9). A putativeARE is also present in the 3′ untranslated region of OsICK4 (AUUUA;nucleotides 729-733 in SEQ ID NO:43). AREs are also present 3′ relativeto the stop codons in OsICK1 (stop codon ‘TGA’: nucleotides 154-156 ofSEQ ID NO:2; ARE ‘ATTTA’: nucleotides 596-600, 600-604, 616-620 and634-638 of SEQ ID NO:2); in ZmICK1 (stop codon ‘TGA’: nucleotides350-352 of SEQ ID NO:5; ARE ‘ATTTA’: nucleotides 545-549 of SEQ ID NO:5)and in PtICK (stop codon ‘TGA’: nucleotides 161-163 of SEQ ID NO:8; ARE‘ATTTA’: nucleotides 284-288 of SEQ ID NO:8). AREs are known to conferinstability to an mRNA (Brewer 1991, Chen and Shiyu 1995, Malter 1989).AREs are furthermore recognized by HuR proteins, i.e., a ubiquitouslyexpressed Elav protein family member.

Binding of HuR to AREs (and the poly-A-tail) enhances transcriptstability (Fan and Steitz 1998). Interestingly, transcripts of themammalian ICK p21Cip1 are stabilized upon UV-C irradiation of cells.Stabilization of p21Cip1 mRNA implies a HuR and increases p21Cip1protein levels (Wang et al. 2000). p21Cip1 is known to enforce S-phasearrest upon the occurrence of DNA damage, e.g., as a result of UVirradiation (Chen et al. 1995). AREs are the target of Rnase E which isconserved from bacteria to humans. Importantly, mRNAs containing morethan one ARE are cleaved more efficiently by Rnase E (Wennborg et al.1995). Thus, the transcript corresponding to SEQ ID NO:9 may be degradedfaster than the transcript corresponding to SEQ ID NO:1. Without wishingto be bound by theory or mode of action, a double function can beenvisaged for OsICK2: a first function related to cell cycle control andimplying rapid turnover of an unstable transcript and a second functionrelated to differentiation (i.e., cell cycle withdrawal) and/or cellcycle arrest implying a transcript of higher stability. Alternatively,the opposite situation can be envisaged as transcript stability andtranslation efficiency can be positively or negatively correlated(Edwards-Gilbert et al. 1997 for review).

Another possibility for the regulation of OsICK2 function lies in thefact that alternative transcript polyadenylation can, as the skilledartisan will know, be tissue-specific and/or developmentally-specificand/or cell cycle-regulated (Edwards-Gilbert et al. 1997). Notableexamples of cell cycle genes of which the transcripts are alternativelypolyadenylated comprise dihydrofolate reductase (DHFR, required for DNAsynthesis during S-phase and for DNA repair; Noé et al. 1999) andmetazoan cyclin D1 (Kiyokawa et al. 1992, Xiong et al. 1991, Yarden etal. 1995). Alternation in DHFR transcript polyadenylation is moreovercell cycle-regulated (Noé et al. 1999). Changes in polyadenylation siteusage in cyclin D1 occur during zebrafish embryonic development (Yardenet al. 1995). The polyadenylation process itself is also regulated bythe cell cycle: the poly-A-polymerase (PAP) is inhibited byphosphorylation by mitosis-specific Cdc2-cyclin B complexes (Colgan etal. 1996) and PAP activity is increased in cells stimulated to enter thecell cycle (Benz et al. 1977, Coleman et al. 1997, Hauser et al. 1978).It will be clear to the skilled artisan that the above mentionedmechanisms of regulation of gene expression may also apply to OsICK4gene expression.

The amino acid sequences of OsICK2 (SEQ ID NO:10) and OsICK4 (SEQ IDNO:44) were aligned with all full-length plant ICK amino acid sequencesknown in the art (see FIG. 2). The amino acid sequences of OsICK2 (SEQID NO:10) and OsICK4 (SEQ ID NO:44) were also aligned with the partialamino acid sequences of the other plant ICKs identified in the presentinvention: the partial amino acid sequence of OsICK1 (SEQ ID NO: 1,encoded by the nucleotide sequence of SEQ ID NO:2), the partial aminoacid sequence of OsICK3 (SEQ ID NO: 12 encoded by the nucleotidesequence of SEQ ID NO:3), the partial amino acid sequence of the Zeamays ICK1 (SEQ ID NO:14 encoded by the nucleotide sequence of SEQ IDNO:5), the partial amino acid sequence of the Zea mays ICK2 (SEQ IDNO:15 encoded by the nucleotide sequence of SEQ ID NO:6) the partialamino acid sequence of the Sorghum bicolor ICK (SEQ ID NO:16 encoded bythe nucleotide sequence of SEQ ID NO:7) and the partial amino acidsequence of the Pinus taeda ICK (SEQ ID NO:17 encoded by the nucleotidesequence of SEQ ID NO:8) (see FIG. 3).

Identities and similarities between the amino acid sequences of OsICK2(SEQ ID NO:10) and OsICK4 (SEQ ID NO:44) and the amino acid sequences ofother known plant ICKs are set forth in Table 6. The overall identitypercentages between all listed ICKs range from 23 to 51% and thesimilarity percentages between 28 and 59%.

TABLE 6 Percentage of identity (bold) and similarity (italics) betweenthe indicated amino acid sequences. The GAP program belonging to the GCGsoftware (Wisconsin Package version 10.1; Madison, Wisconsin) was usedwith default settings gap weight = 8 and length weight = 2. At1 At2 At3At4 At5 At6 At7 Os2 Os4 Alf Che At1 100 34 31 28 31 30 30 24 28 26 32At2 44 100 30 23 23 35 28 24 23 29 27 At3 37 38 100 46 51 30 34 26 46 4137 At4 38 36 55 100 34 26 26 31 38 39 27 At5 39 31 59 45 100 27 28 24 3839 29 At6 37 46 34 36 34 100 49 30 34 24 30 At7 36 40 44 35 38 56 100 3027 23 29 Os2 31 31 42 35 32 37 37 100 46 30 31 Os4 36 32 53 47 45 41 3752 100 41 26 Alf 28 37 49 50 45 32 30 38 47 100 26 Che 39 37 42 35 38 3738 38 36 34 100 At1-7: Arabidospsis thaliana ICK1-7 Os2/4: Oryza sativaICK2/4 Alf: Medicago sativa (alfalfa) ICK Che: Chenopodium rubrum ICK

Identities and similarities were calculated between the amino acidsequences of the relevant parts of the full-length OsICK2 amino acidsequence (SEQ ID NO: 10) and the full-length OsICK4 amino acid sequence(SEQ ID NO:43) with the partial amino acid sequence of OsICK1 (SEQ IDNO:11), the partial amino acid sequence of OsICK3 (SEQ ID NO:12), thepartial amino acid sequence of the Zea mays ICK1 (SEQ ID NO:14), thepartial amino acid sequence of the Zea mays ICK2 (SEQ ID NO:15), thepartial amino acid sequence of the Sorghum bicolor ICK (SEQ ID NO: 16),and the partial amino acid sequence of the Pinus taeda ICK (SEQ ID NO:17). The results of this calculation are included in Table 7.

TABLE 7 Percentage of identity (bold) and similarity (italics) betweenthe indicated amino acid sequences. The GAP program belonging to the GCGsoftware (Wisconsin Package version 10.1; Madison, Wisconsin) was usedwith default settings gap weight = 8 en length weith = 2. OsICK1 OsICK2OsICK3 OsICK4 ZmICK1 ZmICK2 SbICK PtICK OsICK1 100 50 15 48 52 48 71 55OsICK2 64 100 40 36 64 33 41 38 OsICK3 35 44 100 29 25 24 43 47 OsICK457 42 36 100 37 75 30 56 ZmICK1 62 69 31 46 100 37 35 39 ZmICK2 57 38 3181 41 100 37 50 SbICK 86 46 47 38 44 43 100 42 PtICK 55 49 53 67 49 6346 100

All of the plant ICK protein sequences, including the OsICK2 (SEQ IDNO:10) sequence of the invention, were fed into the PHD secondarystructure prediction software (Rost and Sander (1993) J. Mol. Biol 232,584-599 and Rost, B. and Sander, C. (1994) Proteins 19, 55-72). Theresults of this analysis are summarized in FIG. 4. The conserved ICKmotifs 1, 2 and 3 (SEQ ID NO:18, 19 and 20, respectively) present in allplant ICKs as described supra are not only conserved at the amino acidsequence level but also at the level of the predicted secondarystructure with motif 2 being partially involved in an extended β-sheetand motifs 1 and 3 being (partially) α-helical. When present, motifs 4and 6 (SEQ ID NO:21 and 23, respectively) are predicted to be(partially) α-helical. Motif 5 (SEQ ID NO:22) is, when present,predicted to be either partially α-helical or partially an extendedβ-sheet. Outside the conserved motifs, however, the predicted secondarystructures of all plant ICKs, including the OsICK2 protein (SEQ IDNO:10) of the present invention, are different and unique. Thisobservation is clearly in line with the low overall homologies betweenplant ICKs. The OsICK2 protein, in particular, is characterized byextensive α-helical stretches especially in between motifs 5 and 6 andin between motifs 6 and 4. The OsICK2 region between motifs 4 and 3,furthermore, only contains predicted α-helical segments and no extendedβ-sheets. These characteristics are different from those found in theplant ICKs belonging to the same family as OsICK2, namely alfalfa ICKand Arabidopsis ICKs ICK3, ICK4 and ICK5. PHD secondary prediction wasnot performed for the OsICK4 (SEQ ID NO:44) protein.

The presented sequence and secondary structure data, thus, clearlydistinguish OsICK2 from other plant ICKs and, more specifically, fromplant ICKs belonging to the same family as OsICK2.

Example 6 Expression Analysis of OsICK1, OsICK2 and OsICK4

RNA Isolation

Total RNA was extracted from 100-200 mg of immature seed, leave, root,shoot or fraction enriched apical meristems. Frozen tissues were groundwith liquid nitrogen in an ice cold mortar. Two microliters ofextraction buffer (1M Tris-HCl pH 9, 1% SDS, 5% β-mercaptoethanol) andan equal volume of phenol/chloroform/isoamyl alcohol (PCI 25/24/1) wereadded in the mortar and grinding continued until the paste thawed. Themixture was transferred to a tube and vortexted at 10,000 rpm for 10minutes. The aqueous layer was removed and re-extracted with an equalvolume of PCI. After centrifugation (at 10,000 rpm for 10 minutes) thesupernatant was precipitated at 4° C. for 10 minutes with a 1/10 volumeof 3M sodium acetate and 0.8 volumes of cold isopropanol. The pelletrecovered after centrifugation was washed twice with 70% ethanol/0.1Msodium acetate (pH 5.5) before resuspending in 0.5 ml of H₂O. RNA wasprecipitated by adding an equal volume of 4M LiCl and incubating on iceovernight. The RNA was collected by centrifugation and the pellet washedtwice with 70% ethanol/0.1 M sodium acetate. The pellet was drained anddissolved in approximately 80 μl of water.

cDNA Synthesis

The cDNA synthesis was performed using the “Superscript preamplificationsystem for first strand cDNA synthesis” kit from Gibco BRL. Three μg oftotal RNA were mixed with 0.5 μg of an anchored oligo (dT)₂₅ and 12 μlof DEPC water. The mixture was incubated at 70° C. for 10 minutes andthen incubated on ice for at least 1 minute. The following solutionswere then added: 2 μl of 10× PCR buffer, 2 μl of 25 mM MgCl₂, 1 μl of 10mM dNTP mix, and 2 μl of 0.1 M DTT. After an incubation of the mixtureat 42° C. for 5 minutes, 1 μl of Superscript II RT was added and theincubation at 42° C. was continues for 50 minutes. The reaction wasterminated at 70° C. for 15 minutes and then chilled on ice. One μl ofRNase H was added and the reaction was incubated for 20 minutes at 37°C.

Amplification of the Target cDNA

The first strand cDNA obtained as described in the previous paragraphwas amplified using PCR, using the following reactants:

10X PCR buffer 5 μl 2 mM dNTP mix 5 μl sense primer (10 μM) 1 μlantisense primer (10 μM) 1 μl Taq DNA polymerase (Boerhinger) 0.5 μlcDNA 2 μl water 35.5 μl

The primers used for the RT-PCR amplifications were as described inExample 2: primers with SEQ ID NOs: 25 and 26 for OsICK1; primers withSEQ ID NOs: 27 and 28 for OsICK2; and primers with SEQ ID NOs: 31 and 32for OsICK4.

The DNA amplification was carried out using the following conditions(for OsICK4 amplification, the primer annealing temperature was 58° C.instead of 55° C.):

$ {{ {{94{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 3\mspace{14mu}\min}\begin{matrix}{94{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 45\mspace{14mu}\sec} \\{55{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 45\mspace{14mu}\sec}\end{matrix}} \rbrack\mspace{14mu} 15\mspace{14mu}{cycles}\mspace{14mu}{for}\mspace{14mu}{immature}\mspace{14mu}{seed}}\mspace{11mu}{72{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 1\mspace{14mu}\min}\begin{matrix}{94{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 45\mspace{14mu}\sec} \\{55{^\circ}\mspace{14mu}{C.\mspace{14mu}{for}}\mspace{14mu} 45\mspace{14mu}\sec}\end{matrix}} \rbrack\mspace{14mu} 20\mspace{14mu}{cycles}\mspace{14mu}{for}\mspace{14mu}{other}\mspace{14mu}{tissues}$72^(∘)  C.  for  1  min  72^(∘)  C.  for  5  min Hybridization Analysis

Fifty μl of the above-described PCR reaction were loaded on an 1.5%agarose gel. The DNA was blotted onto nylon membranes (Hybond N+,Amersham) and the membranes were baked at 80° C. for 2 hours. Thehybridization was performed using the “North2South ChemiluminescentNucleic Acid Hybridization and Detection” kit from Pierce. The DNA wasprehybridized for 4 hours at 55° C. in the hybridization buffer suppliedby the kit and then hybridized overnight in the same buffer with abiotin-labelled OsICK2 or OsICK1 insert using the North2South BiotinRandom Prime Kit (Pierce).

The membranes were washed twice for 20 minutes at room temperature in2×SSC/G. 1% SDS and twice in 0.5×SSC/0.1% SDS for 15 minutes at 55° C.(at 60° C. in the case of OsICK4). The probe detection and substratedevelopment were performed according to the manufacturer's instructions.

Probe Detection and Substrate Development

The probe detection and substrate development were performed accordingto the manufacturer's instructions. Briefly, after washing the membraneswere blocked with the North2South Blocking buffer for 15 minutes at roomtemperature (RT). The Streptavidin-HRP conjugate was added to theNorth2South Blocking buffer at a 1:300 final dilution and incubated for15 minutes at room temperature. The membranes were washed four times for5 minutes each with North2South wash buffer at RT and then transferredin the North2South Substrate Equilibration buffer for 5 minutes at RT.The substrate development was carried out by covering the membranes witha mix of luminol/enhancer sotution-peroxyde solution for 10 minutes.

The membranes were placed into a film cassette and exposed to film for 1minute. The film was developed by incubation for 5 minutes in thedeveloper and for 5 minutes in the fixer solution.

Results

OsICK1 is predominantly expressed in stems. Lower levels of OsICK1expression are apparent in roots, stem meristems, and seeds, whereas noor only very low expression of OsICK1 could be observed in the leaves(see FIG. 6). OsICK2 is expressed in all rice plant tissues investigatedincluding the leaves (FIG. 6). OsICK4 is also expressed in all riceplant tissues investigated with a lower expression in roots and a higherexpression in leaves and seeds (FIG. 6).

Expression of OsICK1 rises very early during seed development with apeak at 2 days after pollination (DAP) and then slowly declines as seeddevelopment progresses. OsICK2 transcripts are abundantly present inseeds throughout their development with a peak at 8 DAP (see FIG. 7).OsICK4 transcript levels are highly abundant in unpollinated flowers andduring the first 3 days of seed development (i.e., the first 3 daysafter pollination). Thereafter, OsICK4 expression gradually declinesduring the remaining period of seed development (see FIG. 7).

Example 7 In Situ Hybridization of OsICK2 in Rice Immature Seeds

In situ hybridization was performed according to the protocol of deAlmeida-Engler et al. (2000) (Methods, in press) with slightmodifications. Fixation was performed using 4% formaldehyde, 2.5%glutaraldehyde in 0.1 M cacodilate buffer and a 1 hour incubation undervacuum. The fixative was refreshed and treatment continued for 4-5 hoursup to overnight at 4° C. The samples were then dehydrated using ethanol,transferred to xyleen and embedded in paraplast. Subsequently, 10 μmsections were cut and placed on Vectabond coated slides. In vitrotranscription and radioactive labelling (S³⁵) was performed using atranscription kit from Boehringer-Manheim according to themanufacturer's instructions. For this OsICK2 was cloned in a pSP6 vector(Roche Diagnostic) in sense and antisense orientation behind the T7promoter. Hybridization and developing of the slides were performedaccording to de Almeida-Engler et al., supra.

In situ hybridization analysis of OsICK2 expression in rice seedsrevealed that OsICK2 transcripts are spatially confined to the celllayers between the seed coat and the developing endosperm and display apatchy pattern in the developing embryo in seeds collected at 7 DAP(FIG. 8). In seeds collected at 20 DAP, OsICK2 expression is stillstrong in the cell layers between the seed coat and the developingendosperm, as well as in the scutellum (FIG. 9).

Example 8 Transformation of Arabidopsis thaliana with A. thaliana ICKsICK2, ICK3 and ICK4

The full-length coding regions of the A. thaliana ICKs ICK2, ICK3 andICK4 were amplified by polymerase chain reaction using the appropriateprimers introducing different restriction sites. The amplified fragmentswere digested with the corresponding restriction enzymes and cloned inthe corresponding sites of pH35S (Hemerly et al. (1995) EMBO J. 14,3925-3936) containing the CaMV35S promoter and NOS terminator. Theexpression cassettes were subcloned in pGSV4 (Hérouart et al. (1994)Plant Physiol 104, 873-880). The resulting vectors were mobilized by thehelper plasmid pRK2013 into Agrobacterium tumefaciens C58C1Rif^(R)harboring plasmid pMP90. A. thaliana plants of the Col-0 ecotype weretransformed by the floral dip method (Clough and Bent (1998) Plant J.16, 735-743). Transgenic plants were obtained on kanamycin-containingmedia and later transferred to soil for optimal seed production. A totalof 39 and 5 transgenic A. thaliana lines were generated from thetransformations with ICK2 and ICK3, respectively.

Example 9 Transgenic Rice Plants Generated with Different ExpressionPatterns for OsICK4

A number of different constructs have been produced to eitheroverexpress or down-regulate OsICK4 expression. The binary vectorbackbone is pCAMBIA1301. Constructs for modulating OsICK4 expression areoutlined below.

OsICK4 Overexpression

The binary vector backbone is a proprietary vector, pCDV3 (pCAMBIA1301derivative), into which the Gateway system (Life Technologies, Inc) hasbeen introduced. Gateway primers have been designed to amplify OsICK4following the instructions of the manufacturer. The construct foroverexpression of OsICK4 under the control of the GOS2 promoter (dePater et al. (1992) Plant J. 2, 837-844) is designated p0428 ICK4 and isdepicted in FIG. 12.

OsICK4 Co-Suppression

To down-regulate gene expression, an inverted repeat conformation with ahairpin structure has been produced. The two inverted fragments fromOsICK4 are separated by a matrix attachment region (MAR) from Nicotianatabacum (GenBank accession number U67919). The MAR fragment of 315 bphas been PCR-amplified from tobacco genomic DNA using primers sense5′-CGTTGTCAATATCCTGGAAATTTTGC-3′ (SEQ ID NO:35) and antisense5′-CTGCCATTCTTTAGAGGGGATGCTTG-3′ (SEQ ID NO:36), and blunt-end subclonedinto a SmaI-CIP pUC18.

A first OsICK4 fragment (893 bp) cut out from pAD-OsICK4 (full length)with Ecl136II-HincII is blunt ligated into pUC18-MAR cut with Ecl136 II(blunt). The orientation in which Ecl136 II has been restored is chosento allow the excision of the whole inverted repeat (IR) cassette in alater stage. The second OsICK4 fragment (921 bp) is cut out frompAD-OsICK4 (full length) with PstI-HincII (Ecl136II is included in thesequence) and subloned into pUC18-Mars-OsICK4 restricted withPstI-HincII in a sticky-blunt ligation. The vector pUC18-Mars-OsICK4 isdepicted in FIG. 13. The final inverted repeat cassette (2,129 bp;termed OsICK4 IR) can be taken out using Ecl136II for subcloning in, forexample, a plant transformation vector.

OsICK4 IR with GOS2 Promoter

The pCAMBIA vector 1301 containing a 35SCaMV promoter driving GUSexpression has been restricted with XbaI and NcoI to replace the 35SCaMVpromoter with the GOS2 promoter XbaI-NcoI fragment from rice (de Pateret al. (1992) Plant J. 2(6): 837-844).

This vector is then restricted with NcoI and PmlI, and these sitesfilled-in to produce blunt ends. The final inverted repeat cassette(OsICK4 IR) from pUC18 is taken out using Ecl136II and ligated into thebinary vector. The resulting vector is termed p0490 and depicted in FIG.14.

OsICK4 IR with Prolamine Promoter

The 13 kDa prolamine promoter (654 bp) from rice (Wu et al. (1998) PlantCell Physiol. 39:885) has been PCR-amplified using primers sense5′-GAATTCCTTCTACATCGGCTTAGGTGTAGC-3′ (SEQ ID NO:46) and antisense5′-CCATGGTGTTGTTGGATTCTACTACTATGC-3′ (SEQ ID NO:47) and the subsp.Japonica genomic DNA. The fragment was further subcloned in pUC18 SmaICIP. An XbaI and NcoI restriction produced a promoter fragment that wassubcloned into the pCAMBIA 1301 35SCaMV-GUS vector also restricted withXbaI and NcoI. This led to the replacement of the CaMV promoter by theprolamine promoter. This prolamine-GUS binary vector was then restrictedwith NcoI and PmlI to introduce the IR cassette. After filling-in thesesites to produce blunt ends, the Ecl136II final IR cassette (OsICK4 IR)was taken from the pUC18 and ligated into the binary vector. Theresulting vector is termed p0489 and depicted in FIG. 15.

OsICK4 IR with Oleosin Promoter

The 18 kDa oleosin promoter (1236 bp) from rice (Wu et al. (1998) PlantCell Physiol 39:885) has been PCR-amplified using primers sense5′-GAACAAAGATGGTCAGCCAATACATTGATC-3′ (SEQ ID NO:48) and antisense5′-GGCCATGGCTAAGCTAGCTAGCAAGATGAA-3′ (SEQ ID NO:49) and the subsp.Indica genomic DNA. The fragment was further subcloned into pUC18 SmaICIP. An XbaI and NcoI restriction produced a promoter fragment that wassubcloned into the pCAMBIA 1301 35SCaMV-GUS vector also restricted withXbaI and NcoI. This led to the replacement of the CaMV promoter by theoleosin promoter. This oleosin-GUS binary vector was then restrictedwith NcoI and PmlI to introduce the IR cassette. After filling-in thesesites to produce blunt ends, the Ecl136II final IR cassette (OsICK4 IR)was taken from the pUC18 and ligated into the binary vector. Theresulting vector is termed p0488 and depicted in FIG. 16.

OsICK4 IR with Glutelin Promoter

The glutelin 3A promoter (984 bp) from rice (Wu et al. (1998) J.Biochem. 123: 386) has been PCR-amplified using primers sense5′-AGAAGAAAGATAAATAACCGAAACTATTTG-3′ (SEQ ID NO:50) and antisense5′-GGACATGTTTTTGTGGGACTGAACTCAATG-3′ (SEQ ID NO:51) and the subsp.Indica genomic DNA. The amplified fragment was further subcloned intopUC18 SmaI CIP. A SmaI and AflIII restriction produced a promoterfragment that was subcloned into the pCAMBIA 1301 35SCaMV-GUS vectorrestricted with SmaI and NcoI. This led to the replacement of the CAMVpromoter by the glutelin promoter. This glutelin-GUS binary vector wasthen restricted with BglII and PmlI to introduce the IR cassette. Afterfilling-in these sites to produce blunt ends, the Ecl136II final IRcassette (OsICK4 IR) was taken from the pUC18 and ligated into thebinary vector. The resulting vector is termed p0559 and depicted in FIG.17.

Example 10 Agrobacterium-Mediated Rice Transformation

Mature dry seeds of the rice japonica cultivars Nipponbare or Taipei 309are dehusked, sterilised and germinated on a medium containing 2,4-D(2,4-dichlorophenoxyacetic acid). After incubation in the dark for fourweeks, embryogenic, scutellum-derived calli are excised and propagatedon the same medium. Selected embryogenic callus is then co-cultivatedwith Agrobacterium. Widely used Agrobacterium strains such as LBA4404 orC58 harbouring binary T-DNA vectors can be used. The hpt gene incombination with hygromycin is suitable as a selectable marker systembut other systems can be used. Co-cultivated callus is grown on2,4-D-containing medium for 4 to 5 weeks in the dark in the presence ofa suitable concentration of the selective agent. During this period,rapidly growing resistant callus islands develop. After transfer of thismaterial to a medium with a reduced concentration of 2,4-D andincubation in the light, the embryogenic potential is released andshoots develop in the next four to five weeks. Shoots are excised fromthe callus and incubated for one week on an auxin-containing medium fromwhich they can be transferred to the soil. Hardened shoots are grownunder high humidity and short days in a phytotron. Seeds can beharvested three to five months after transplanting. The method yieldssingle locus transformants at a rate of over 50% (Aldernita and Hodges(1996), Chan et al. 1993, Hiei et al. 1994).

Example 11 Two-Hybrid Interaction of OsICK2 and OsICK4 with CDC2-Os1 andCYCD3Os

The interactions of OsICK2 and OsICK4 with Cdc2-Os1 and CycD3-Os (therice cyclin D3) were verified using a yeast two hybrid approach. In apreliminary step, yeast strain PJ69-4A (MATa trp1-901 leu2-3,112 ura3-52his3-200 gal4(deleted) gal80(deleted) LYS2::GAL1-HIS3 GAL2-ADE2met2::GAL7-lacZ) was transformed with Cdc2-Os1 in bait vector pBDGal4(Stratagene), using the lithium acetate method, well known to thoseskilled in the art. The rice Cdc2-Os1 was previously obtained by PCR asdescribed in Example 1. The Cdc2-Os1 was also cloned in the pADGal4 preyvector. The junctions between the pBDGal4 binding domain and the pADGal4activation domain and the Cdc2-Os1 fragment were sequenced to verifycorrect in-frame fusion.

The full-length OsICK2 and OsICK4 cDNA clones in the pADGal4 vector wereobtained as described in Examples 3 and 4. These cDNAs were also clonedin the pBDGal4 vector. The public databases were screened for conservedmotifs found in all D-type cyclins. This search yielded a hit consistingof a rice EST clone (GenBank accession number AU082424). PCR wasperformed to amplify a part of this clone using the following primers:antisense 5′-ACTCCTTGTCCCTATCGACACACC-3′ (SEQ ID NO:52) and sense5′-CCATGGGGGACGCCTCGGCATCCA-3′ (SEQ ID NO:53). The amplified fragmentwas cloned in pUC18 and used as a radioactive probe for the two-hybridcDNA library screening.

Approximately 750,000 plaque-forming units from the cell suspensiontwo-hybrid cDNA library were screened. Plaques were transferred toHybond N+ membranes (Amersham). The filters obtained were prehybridizedin sodium phosphate 0.25M, (pH 7.2), SDS 7% at 60° C. for 4 hours.Hybridization was performed with the prehybridization buffer containing50 ng of [α]³²P-dCTP-labelled probe at 60° C. overnight (protocol ofChurch G. M. and Gilbert W. PNAS USA 81:1991-1994). The filters werewashed twice with 1×SSC, 0.1% SDS at 60° C. for 30 minutes and then oncewith 0.1×SSC, 0.1% SDS at 60° C. for 30 minutes. The membranes wereplaced into a film cassette and exposed to film for 6 hours.

Two putative positive clones were identified. A second round ofscreening on these clones was performed. Pure positive plaques wereisolated and phagemids were excised therefrom. Sequencing of the twoclones revealed that the clones were identical with a full length sizeof 1.7 kb. The clones contain an ORF of 1140 bp encoding a protein of380 amino acids. This protein is designated cyclin D3 from rice(CycD3-Os). The yeast stain PJ69-4a containing the bait vector pBDGal4with Cdc2-Os1 was retransformed with the prey vector pADGal4 containingthe in-frame OsICK2 or CycD3-Os, and plated on selective Leu-Trp-His- toselect for growth. The yeast expressing both proteins could effectivelygrow, illustrating the interaction of Cdc2-Os1 with OsICK2 and CycD3-Os,respectively.

As positive controls, growth was assessed in yeast containing the preyvector pADGal4 with Cdc2-Os1 and retransformed with the bait vectorpBDGal4 containing OsICK2 or CycD3-Os. In all cases, yeast growth didoccur. Appropriate negative controls were incorporated in which any ofthe described bait pBDGal4 or prey pADGal4 vectors containing eitherCdc2-Os1, OsICK2 or CycD3-Os were combined with the empty prey pADGal4or pBDGal4 vectors, respectively. None of these combinations was able tosustain yeast growth on selective medium.

From the molecular cloning of OsICK4 (see Example 4), it is evident thatOsICK4 interacts physically with Cdc2-Os1.

In further experiments, the interaction between OsICK2 and OsICK4 withCycD3-Os, respectively, was assessed. Both OsICK2 and OsICK4 were shownto interact with CycD3-Os in a yeast two-hybrid system as describedabove.

Example 13 Expression of Recombinant ICK Proteins in Bacterial Cells

In this example, the ICK molecules of the present invention areexpressed as a recombinant glutathione-S-transferase (GST) fusionpolypeptide in E. coli and the fusion polypeptide is isolated andcharacterized. Specifically, ICK molecules are fused to GST and thisfusion polypeptide is expressed in E. coli, e.g., strain PEB199.Expression of the GST-ICK fusion protein in PEB199 is induced with IPTG.The recombinant fusion polypeptide is purified from crude bacteriallysates of the induced PEB199 strain by affinity chromatography onglutathione beads. Using polyacrylamide gel electrophoretic analysis ofthe polypeptide purified from the bacterial lysates, the molecularweight of the resultant fusion polypeptide is determined.

Example 14 Isolation of the Rice OsICK5 Genomic Clone and Prediction ofthe OsICK5 cDNA and Protein Sequences

The public databases were screened for the “GRYEW” amino acid motiflocated at the carboxy-termini of all ICKs. This research yielded a hitwith a non annotated HTGS full length clone (GenBank accession numberAP003525). PCR was performed to amplify this genomic clone using thefollowing primers:

-   -sense:5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACAATGGGGAAGAA    GAAGAAGCGCGACG-3′ (SEQ ID NO:57); and-   -antisense:5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTCAGCCGCTGCCCAC CGCGG-3′    (SEQ ID NO:58).    The amplified fragment was cloned in the pDON201 (Gateway system;    Life Technologies).    Said sense and antisense primers (SEQ ID NO:57 and SEQ ID NO:58,    respectively) are subsequently used to isolate the full-length    OsICK5 cDNA by RT-PCR (see, e.g., Example 6).    The isolated OsICK5 genomic clone (SEQ ID NO:56; FIG. 19) is part of    the HTGS clone with GenBank accession number AP003525 (version 1)    but no open reading frame or protein annotation is presented.    Therefor, the open reading frame and protein deduced thereof were    predicted. The predicted full-length OsICK5 cDNA is defined by SEQ    ID NO:54 and the OsICK5 protein sequence deduced thereof is defined    by SEQ ID NO:55. During the prediction process, difficulties araised    in defining the correct exon-intron borders. These uncertainties are    reflected in the OsICK5 cDNA sequence (SEQ ID NO:54; FIG. 20A) by    two ‘N’ nucleotides (i.e. may be any nucleotide (A, T, C or G) or a    stretch of such nucleotide residues) at the positions 355 and 356 of    SEQ ID NO:54. As defined in SEQ ID NO:54, the stretch of uncertain    nucleotides may have a length of zero to 265 nucleotides, i.e. the    maximum length between the intron involved. Said uncertain    nucleotides being translated into an uncertain amino acid residue X    (i.e. may be any amino acid residue or a stretch of such amino acid    residues) at the position 119 of SEQ ID NO:55. As defined in SEQ ID    NO:55, the stretch of uncertain amino acids may have a length of    zero to 88 amino acids, i.e. the maximum protein sequence deducable    from the whole intron involved.    Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for downregulating the activity of a cyclin-dependent kinaseinhibitor (ICK) in a monocot plant selected from the group consisting ofrice, maize, wheat, barley, sorghum, oats, sugarcane, rye and triticale,said method comprising introducing into the monocot plant an antisensemolecule about 20 nucleotides in length and targeted to a nucleic acidmolecule encoding an ICK, said nucleic acid molecule encoding an ICKselected from the group consisting of SEQ ID NOs: 4, 43, and 45, or anucleic acid molecule at least 95% identical thereto.
 2. The method ofclaim 1 wherein plant growth is enhanced, senescence of the plant isdelayed, seed yield and/or seed size is increased, or enhanced formationof lateral organs from the plant tissue and/or an organ is obtained. 3.A method for improving tolerance to an environmental stress condition ina monocot plant selected from the group consisting of rice, maize,wheat, barley, sorghum, oats, sugarcane, rye and triticale, said methodcomprising introducing into the plant an antisense molecule about 20nucleotides in length and targeted to a nucleic acid molecule encoding acyclin-dependent kinase inhibitor (ICK), said nucleic acid moleculeencoding an ICK selected from the group consisting of SEQ ID NOs: 4, 43,and 45, or a nucleic acid molecule at least 95% identical thereto andwherein the environmental stress condition is selected from the groupconsisting of drought stress, salt stress, temperature stress, andnutrient deprivation.
 4. A method for increasing seed number in amonocot plant selected from the group consisting of rice, maize, wheat,barley, sorghum, oats, sugarcane, rye and triticale, said methodcomprising introducing into the plant an antisense molecule about 20nucleotides in length and targeted to a nucleic acid molecule encoding acyclin dependent kinase inhibitor (ICK) selected from the groupconsisting of SEQ ID NOs: 4, 43, and 45, or a nucleic acid molecule atleast 95% identical thereto, wherein the antisense molecule is under thecontrol of an endosperm-specific promoter.
 5. The method of claim 4wherein the endosperm-specific promoter is the prolamin promoter.
 6. Themethod of claim 4 wherein the plant is rice.